User:Jsilke/Aza

Introduction
[Shellfish toxins] are naturally occurring chemicals that can contaminate certain filter feeding bivalve molluscs primarily. The shellfish contaminated with these toxins frequently look, smell, and taste normal, however, when humans eat such seafood, a variety of illnesses can result. One of these toxin groups, the Azaspiracids (AZAs)were first detected in mussels (Mytilus edulis) in Ireland in 1995. AZAs have been identified in a number of other countries since then and in other bivalve species, including oysters (Crassostrea gigas, Ostrea edulis), scallops (Pecten maximus), clams (Tapes phillipinarium), cockles (Cardium edule), and razor fish (Ensis siliqua) Since their discovery this toxin has been responsible almost annually for the closure of many shellfish growing areas for much of the season, in order to protect consumers’ health, particularly in [Ireland]. Human intoxication, termed “Azaspiracid poisoning” (AZP), associated with the consumption of shellfish contaminated with AZAs, has been recorded, however, toxicological data on the effects of AZAs are limited. Observations in humans and in animal studies, as well as in vitro studies report the ability of AZA to affect the gastrointestinal tract and to cause profound biological perturbations at the cellular level. Azaspiracid is not an individual compound; it is a group of structurally similar [polyethers] with some novel structural features. These toxins accumulate in bivalve molluscs that feed on toxic planktonic microalgae. A species of phytoplankton (Azadinium spinosum) responsible for producing AZA has recently been isolated in the North Sea and it is likely that this is a widespread organism that has been missed in routine monitoring due to its small size. In laboratory experiments, azaspiracids can induce widespread organ damage in animals and because of that they are probably more dangerous than previously known classes of shellfish toxins. Azaspiracids differ from any of the previously known toxins found in shellfish or dinoflagellates.

Azaspiracid Shellfish Poisoning (AZP)
In November 1995, at least eight people in the Netherlands became ill after eating mussels (Mytilus edulis) cultivated at Killary Harbour, Ireland. Although the symptoms resembled those of diarrhetic shellfish poisoning (DSP), concentrations of the major DSP toxins were very low [1]. No known organisms producing DSP toxins were observed in water samples collected at that time. In addition, a slowly progressing paralysis was observed in the mouse assay using the mussel extracts. These neurotoxic symptoms were quite different from typical DSP toxicity [2]. Subsequently azaspiracid was identified and the new toxic syndrome was called azaspiracid shellfish poisoning (AZP) [3]. In vivo studies with mice were carried out to elucidate the pathological injuries cause by the toxin. By per os administration, the toxin caused necrosis in the lamina propria of the small intestine and in lymphoid tissues such as thymus, spleen and the Peyer's patches. Both T and B lymphocytes were injured. Additionally a fatty change was observed in the liver. These injuries distinctly differed from those caused by the representative diarrhetic shellfish toxin, okadaic acid [2].

Azaspiracid Sources
AZP is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis). The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g, µg/g) recorded to-date were in mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks also indicates these could pose a threat to human health [4].

Identification of Azaspiracids in Europe
Although first incidents of human intoxications by azaspiracid discovered in Ireland, the search for the azaspiracid toxins, in other European countries has led to the discovery of these toxins in shellfish from France and Spain [5]. Azaspiracid contamination of several types of bivalve shellfish species has now been confirmed throughout the western coastline of Europe [6, 7]. Azaspiracids were identified for example in mussels (Mytilus galloprovincialis), 0.24 µg/g, from Galicia, Spain, and scallops (Pecten maximus), 0.32 µg/g, from Brittany, France. Toxin profiles were similar to those found in the equivalent shellfish in Ireland in which AZA1 was the predominant toxin.

Toxic Dose Assessment for AZP
The lowest-observable-adverse-effect-level (LOAEL) of AZA has been estimated. Ofuji et al. [8] reported a level for total AZAs in raw mussel meat in poisoning incidents of 1.4 µg/g of meat. At a consumption of 100 to 300 g per meal, this corresponds to an intake of 140 to 420 µg AZAs/person. A no-observable-adverse-effect-level (NOAEL) is often estimated at one-tenth the LOAEL and is used to estimate the maximum permitted levels in food.

Thus, the NOAEL would be 14 to 42 µg per person (consumption of 100 to 300 g shellfish meat/meal). As a consequence the maximum allowance concentration in shellfish meat would be 14 µg/100 g or 140 µg/kg. (Note that the usual factor of 10 was not applied to the NOAEL to allow for intraspecies differences in human population.) A comparison of toxin limits in foods of the European Parliament 2004 shows the limits for marine toxins in foods are Paralytic Shellfish Poison (PSP), 800 µg/kg; Amnesic Shellfish Poison (ASP), 20 µg of domoic acid/kg; for okadaic acid, dinophysitoxins and pectenotoxins together, 160 µg of okadaic acid equivalents/kg; for yessotoxins 1 mg/kg; and for AZAs 160 µg/kg. (www.europarl.eu.int/commonpositions/2004/[df/c5-0009-04-part.2_en.pdf)

Chemistry of Azaspiracid
Azaspiracids (AZAs) differ structurally from any of the previously known marine toxins, e.g., yessotoxins, pectenotoxins, maitotoxins, palytoxin, pinnatoxin, gymnodimine and the spirolides [9]. AZAs have unique spiro ring assemblies, a cyclic amine instead of a cyclic imine group and a carbocyclic or lactone ring is absent [8]. At present five AZAs are known (Figure 1). Ofuji et al. [8] identified azaspiracid-2 (AZA-2) and azaspiracid-3 (AZA-3) and demonstrated that these compounds were 8-methylazaspiracid and 22-demethylazaspiracid, respectively. Lastly the same authors [10] determined the structure of two further analogues of azaspiracid found in mussels namely azaspiracid-4 (AZA-4) and azaspiracid-5 (AZA-5) and showed that these compounds were 3-hydroxy-22-demethylazaspiracid and 23-hydroxy-22-demethylazaspiracid, respectively. Recently, five new hydroxyl analogs of azaspiracids (AZA-6--11) in shellfish have been determined using liquid chromatography with multiple tandem mass spectrometry [11]. During an instrumental validation study for rapid quantification of AZA-1 in complex biological matrix, a new azaspiracid analog (AZA-7c) was discovered [12]. It is not currently known how or why AZAs are produced and if they are products of metabolism or are liable to structural modification within shellfish. By analogy with pectenotoxins and yessotoxins, which undergo structural modification by hydroxylation in mussels, it is likely that AZA-4 and AZA-5 are oxidized metabolites of AZA-3. Hence, AZA, AZA-2 and AZA-3 are likely to be the genuine products of a marine organism. Azaspiracid was considered as the major causative agent in 1998[13]. What is interesting is these authors also reported that mussel extracts did not show a significant decrease of toxicity when the extract was heated at 50 °C for 150 minutes in 1 M acetic acid/methanol or 1 M ammonium hydroxide solution and no significant change in toxicity occurred in solution during storage. Therefore AZAs are assumed to be relatively stable compounds.

Chemical Assays of Azaspiracid
The first LC-MS quantitative determination method reported for AZAs was based on selected ion monitoring (SIM) detection [8], with one ion per compound and external calibration. Linearity was checked over a relatively wide concentration range (50 pg to 100 ng). The LC-MS method for AZAs was presented by James et al. [3] and a micro liquid chromatography-tandem mass spectrometry method (micro-LC-MS-MS) was developed for the determination of AZAs by Draisci et al. [14]. Lehane et al. [15] reported the development of an LC-ESI-MS method for the determination of the three most prevalent AZA toxins (AZA1-3), as well as the hydroxylated analogues (AZA4-5). Using a triple-quadrupole mass spectrometer, ultra-high sensitive method was developed with the low-femtogram level of AZAs on-column [12].

Toxicology of Azaspiracid
Gastrointestinal illnesses are observed in AZP episodes, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far AZA’s mechanism of action is largely unknown. It is a unique toxin group that targets the liver, lung, pancreas, thymus, spleen (T and B-lymphocytes) and digestive tract.

Initial investigations have shown that AZA-1 is cytotoxic to a range of cell types, and cytotoxic effects are both time- and concentration-dependent. However, AZA-1 took an unusually long time (>24 h) to cause complete cytotoxicity in most cell types [16] AZA-1 did not inhibit protein phosphatase 2A. In vitro studies performed in human cells from healthy donors suggest that the threshold for AZA analogues to modify cellular function would be 24 mg/kg for a 60 kg person. AZA-4 appeared to be a novel inhibitor of plasma membrane Ca2+ channels, affecting at least store-operated channels in Ca signaling, showing an effect clearly different from other AZA analogs [17]. Recently Colman et al. [18] demonstrated that AZA-1 is a potent teratogen to finfish. Satake et al. [13] reported a lethal dose in mice of purified AZA 200 µg/kg, i.p. The LD50 for AZA to mice at (oral administration) was 500-600 µg/kg [19]. Lethal doses for AZA-2 and -3 in mice were 110 and 140 µg/kg (i.p.), respectively [8] and for AZA-4 and AZA-5 approximately 470 and less than 1000 mg/kg, respectively [10]. Mice exposed to AZA by i.p. injection react differently than those exposed to other shellfish toxins. After i.p. dosing of AZA to male mice, the animals became sluggish, sat still in the corners and showed progressive paralysis and labored breathing. No diarrhea was observed. At low doses the animals died two to three days after dosing. The minimal lethal dose for mice was reported to be 150 µg/kg [13]. SUMMARY

Azaspiracid is a potent toxic natural product that represents a human health hazard due to its seasonal contamination of mussels (Mytilus edulis) that have been cultivated for human consumption. This unique toxin was isolated recently from Irish mussels and is responsible for the acute poisoning of several humans. Azaspiracid is not an individual compound; it is a group of structurally similar polyethers with unprecedented structural features and differs from any of the previously known toxins found in shellfish or dinoflagellates. In laboratory animals, AZAs can induce widespread organ damage as well as neurotoxic effects including progressive paralysis. They may be more dangerous than previously known classes of shellfish toxins. Footnotes to Introduction paragraph:

1.	The European Union, aware of the danger incurred in eating certain fish products, has issued a set of hygiene and health directives for the purpose of preventing disease and safeguarding consumer health. In particular, directive 91/492/EEC, of 15 July 1991, lays down the sanitary norms applicable to the production and commercialization of live bivalve molluscs, echinoderms, tunicates and marine gastropods and regulates the whole system involving these products from their origin to consumption. More recently, through Commission Decision dated 15 March 2002 (EC OJ 175/62 of 16.3.2002) the EU has set new standards for the implementation of directive 91/492/EEC with respect to the maximum levels and analysis methods for some marine biotoxins. 2.	Protoperidinium crassipes, Class Dinoflagellata, firstly described by Kofoid CA. Dinoflagellata of the San Diego Region, III. Description of new species. Univ. Calif. Publ. Zool. 3: 299-304. 1907, present description: Balech E. El genero Protoperidinium Bergh 1881 (Peridinium Ehrenberg 1831, partim). Rev. Mus. Argentina