PART II ASSESSMENT OF THE RISK OF BIOTOXINS IN BIVALVE MOLLUSCS FTP551_Book.indb 31 27/03/2012 15:34:03 33 Azaspiracids This chapter was compiled by: Prof Michael Ryan, University of Dublin, Ireland Dr Emiko Ito, Chiba University, Japan Dr Gerrit Speijers, National Institute of Public Health and the Environment, the Netherlands Dr Philipp Hess, Marine Institute, Ireland 1. BACKGROUND INFORMATION1 The syndrome that later was named azaspiracid poisoning (AZP) was detected for the first time in 1995 among consumers in the Netherlands after eating blue mussels from Ireland. The symptoms were similar to those of diarrhoeic shellfish poisoning (DSP), but the concentration of the DSP toxins was low. Subsequently, the azaspiracid (AZA) toxin group was discovered. Thus far, AZAs have only been detected in Europe. The European Union (EU) has set a regulatory level of 0.16 mg/kg with mouse bioassay (MBA) as the reference method. However, an MBA protocol with adequate specificity or detectability has not been validated. Current testing is based on preliminary liquid chromatography with mass spectrometry detection (LC-MS) methods using a limited supply of AZA-1 reference standard. 2. ORIGINS AND CHEMICAL DATA Azaspiracids are nitrogen-containing polyether toxins with a unique spiral ring assembly, a cyclic amine and a carboxylic acid, and were first detected in mussels (Mytilus edulis) in Ireland in 1995. Currently, 20 different congeners have been identified (Satake et al., 1998b; Ofuji et al., 1999a, 2001, James et al., 2003, Rehmann, Hess and Quilliam, 2008); however, toxicological information is only available for AZA- to -5 (Satake et al., 1998b; Ito et al., 1998, 2000, 2002; Ofuji et al., 1999a, 2001). Compared with the other analogues, AZA-1, -2 and -3 are the major contributors to the overall toxic equivalents found in shellfish, both because of their high concentration and because of their comparatively high toxicity (Figure 1). Recently, AZA-1, -2 and -3 were identified within the heterotrophic dinoflagellate, Protoperidinium spp. (Yasumoto, 2001). However, Krock et al. (2009) and Krock, Tillmann and Cembella (2009) found a small dinoflagellate in the North Sea to produce AZA in culture, and thus it is presumed that this organism or related species were also responsible for production of AZA in Ireland and other countries where AZAs have been reported. As described by Twiner et al. (2005), AZAs have been detected in bivalve species other than mussels, including oysters (Crassostrea gigas, Ostrea edulis), scallops (Pecten maximus), clams (Tapes phillipinarium), cockles (Cardium edule) and razor fish (Ensis siliqua) (Hess et al., 2003; Furey et al., 2003). Several countries, including France, Ireland, Italy, the Netherlands, Norway, Spain and the United Kingdom of Great Britain and Northern Ireland, have documented either cases of AZA intoxications and/or contaminated shellfish (Satake et al., 1998a). Although the human symptoms resembled those of DSP, the illness was subsequently named AZP (Ofuji et al., 1999a) once AZA was identified as a novel toxin (Satake et al., 1998b). 1 Corresponds to the “Background Information” section of the Expert Consultation Report. FTP551_Book.indb 33 27/03/2012 15:34:03 34 Assessment and management of biotoxin risks in bivalve molluscs FIGURe 1 Azaspiracids: AZA-1 (R1,2,4 = H; R3 = CH3), AZA-2 (R1,4 = H; R2,3 = CH3) Notes: The initial structure proposed by Satake et al. (1998b) was corrected by Nicolaou et al. (2004a, 2004b). The corrected structure is shown. 3. BIOLOGICAL DATA 3.1 Biochemical aspects 3.1.1 Absorption, distribution and excretion/toxicokinetics No data reported. 3.1.2 Biotransformation No information on pathways of AZA metabolism in animals has been reported. 3.1.3 Effects on enzymes and other biochemical parameters/mechanism of action At present, there are virtually no data on the mechanism of action, although based on the studies that Roman et al. (2002) performed, some information on targets on cellular level became available. They report on potential cellular targets of AZA-1, which causes diarrhoeic and neurotoxic symptoms and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F–actin levels and mitochondrial membrane potential. While AZA-1 did not modify mitochondrial activity, it did decrease F–actin concentration. These results indicate that the toxin does not have an apoptotic effect but uses F–actin for some of its effects. Therefore, cytoskeleton seems to be an important cellular target for AZA-1 effect. AZA-1 did not induce any modification in membrane potential, which does not support for neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZA-1 increased cytosolic calcium and cAMP levels, and did not affect pHi. Cytosolic calcium increase seemed to be dependent on both the release of calcium from intracellular Ca2+ increase pools and the influx from extracellular media through Ni2+-blockable channels. AZA-1 induced Ca2+ increase is negatively modulated by agents that regulate protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and cAMP increase. The effect of AZA-1 on cAMP is not extracellularly Ca2+ dependent and unsensitive to OA (Roman et al., 2002). AZAs were cytotoxic to P388 cells but to KB cells the potency was much less prominent (EU/SANCO, 2001). AZA did not inhibit PP2A. It was noted that in vitro FTP551_Book.indb 34 27/03/2012 15:34:04 Assessment of the risk of biotoxins in bivalve molluscs: azaspiracids 35 studies performed in human cells from healthy donors suggest that the threshold for AZA analogues to modify cellular function would be 24 µg/kg for a 60 kg person. 3.1.3.1 Effect of AZA on DNA fragmentation In vivo administration of AZA was studied to see whether it causes deoxyribonucleic acid (DNA) fragmentation. Mice organs (ICR male 4 week) were stained with apoptotic peroxidase in situ apoptosis kit after p.o. treated with AZA. By this, the livers showed apoptosis in all examined cases (300 µg/kg: 1, 2 and 4 hours, and 600 µg/kg: 4, 18 and 24 hours), but not in the lung and kidney. 3.1.3.2 Effect of AZA‑1 on an in vitro model of GI permeability The human colonic cell line Caco-2 was used to assess the impact of AZA-1 on intestinal barrier function. Barrier function was measured by transepithelial electrical resistance (TEER). TEER works by measuring the rate of flux of ions across the paracellular pathway as an electrical resistance. Disruption of the paracellular barrier is a contributing factor to increased fluid secretion in diarrhoea. AZA-1 was found to decrease TEER in a dose-dependent fashion over time. A significant decrease was observed at 5 nM at 24 hours (Ryan, Hess and Ryan, 2004). This functional assay may be developed as a possible in vitro assay for AZA. 3.1.3.3 Cytotoxic and cytoskeletal effects of AZA‑1 on mammalian cell lines Initial investigations have shown that AZA-1 is differentially cytotoxic to several different cell types as determined by the MTS assay, which measures mitochondrial activity. Calculated EC50 values for the Jurkat cell line (lymphocyte T cells) were 3.4, 1.1 and 0.9 nM for 24, 48 and 72 hour exposures, respectively. The effect of AZA-1 on membrane integrity was tested by measuring the release of a cytosolic enzyme, glucose-6-phosphate dehyrogenase (G6PD), from Jurkat cells. Significant elevations in G6PD activity were detected in the extracellular medium for AZA-1 exposed cells, with preliminary EC50 values of 0.2 and 0.07 nM for 24 and 48 hours of exposures. AZA-1 was also reported to be capable of rearranging cellular F-actin in Jurkat cells. This was apparent with the concurrent loss of pseudopodia and cytoplasmic extensions that function in mobility and chemotaxis prior to cytotoxicity (Twiner et al., 2005). 3.2 Toxicological studies 3.2.1 Acute toxicity Oral studies Acute oral studies with AZA in mice were performed. AZA was extracted from mussels collected in Killary Harbour, Ireland, in February 1996. During the course of toxin purification, the major toxin was concentrated in a lipid fraction coded Killary Toxin-3 (KT3) (as cited in Ito et al., 2000). By oral administration (by gavage) of 60 µl of this KT3 fraction, mice did not show any clinical changes during 24 hours. At autopsy after 4 hours, active secretion of fluid from the ileum and debris of necrotizing epithelial cells from upper portion of the villi were observed in the lumen (SEM), and after 8 hours, erosion of the villi from the top resulted in the shortened villi, and prominent accumulation of fluid was observed accompanying edema in the lamina propria. Then, after 24 hours, these changes were not observed but epithelial cells of adjacent villi were fused to each other (Ito et al., 1998). Male ICR mice receiving orally by gavage a single dose of 500, 600 or 700 µg purified AZA/kg b.w. did not show any behavioural changes within 4 hours. Number of survivors after 24 hours were 0/2, 3/6 and 1/2 at 500 (8 weeks old), 600 (5 weeks old) and 700 µg/kg b.w. (5 weeks old), respectively. At 600 and 700 µg/kg b.w., diarrhoea and b.w. decrease were observed within 24 hours. FTP551_Book.indb 35 27/03/2012 15:34:04 36 Assessment and management of biotoxin risks in bivalve molluscs At single oral doses of 300–700 µg/kg b.w., AZA caused dose-dependent changes in small intestines (necrotic atrophy in the lamina propria of the villi) and in lymphoid tissues, such as thymus, spleen and Peyer’s patches.