Toxicon 56 (2010) 842847

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Cytotoxic effect of palytoxin on mussel

M. Carmen Louzao a, Begona Espina a, Eva Cagide a, Isabel R. Ares a, Amparo Alfonso a, Mercedes R. Vieytes b, Luis M. Botana a, *
a b

Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain Departamento de Fisiologia Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain

a r t i c l e i n f o
Article history: Received 1 September 2009 Received in revised form 5 February 2010 Accepted 22 February 2010 Available online 3 March 2010 Keywords: Azaspiracid-1 Cytotoxicity Hepatopancreas Mantle Metabolic rate Mussel Okadaic acid Palytoxin Phycotoxins

a b s t r a c t
Palytoxin is a large and complex polyhydroxylated molecule with potent neurotoxic activity. Dinoagellates from the Ostreopsis genera were demonstrated to be producers of this compound and analogues. Even though initially palytoxin appearance was restricted to tropical areas, the recent occurrence of Ostreopsis outbreaks in Mediterranean Sea point to a worldwide dissemination probably related to climatic change. Those dinoagellates can bioaccumulate in shellsh, especially in lter-feeding mollusks and have been involved in damaging effects in seafood or human toxic outbreaks. The present study describes palytoxins effect on metabolic activity of mantle and hepatopancreas cells from the mussel Mytilus galloprovincialis Lmk. Our results indicate that palytoxin is highly cytotoxic to mussel cells; unlike it happens with other toxins more common in European coasts such as okadaic acid and azaspiracid. These ndings have a special signicance for the marine environment and aquiculture since they are evidence for the ability of palytoxin to affect the integrity of bivalve mollusks that are not adapted to the presence of this toxin. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Palytoxin (PLT) and related compounds are potent neurotoxic phycotoxins rst isolated from zoanthids of the genus Palythoa (Moore and Scheuer, 1971; Ishida et al., 1983). However, benthic microalgae belonging to the genus Ostreopsis have been proposed as their possible biogenetic origin because PLT and its analogues were found in some species (Yasumoto, 1998; Taniyama et al., 2003; Aligizaki et al., 2008; Ciminiello et al., 2008; Louzao et al., 2008; Cagide et al., 2009). These dinoagellates are more common in tropical and subtropical waters but, probably as a result of climatic change, recently started to appear in the Mediterranean Sea (Aligizaki et al., 2008; Ciminiello et al.,
Abbreviations: AZA, Azaspiracid; DSP, Diarrheic shellsh poisoning; OA, Okadaic acid; PLT, Palytoxin. * Corresponding author. Tel./fax: 34 982 252 242. E-mail address: luis.botana@usc.es (L.M. Botana). 0041-0101/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.02.027

2008; Louzao et al., 2008). In this case their blooms have been linked to respiratory irritation in people exposed to seawater spray, especially in Italy, presenting cough, dyspnea, sore throat, rhinorrhea, fever, headache, lacrimation, nausea/vomiting and dermatitis (Durando et al., 2007). It was found that Ostreopsis collected in the Mediterranean Sea produced PLT-like compounds that are highly toxic and functionally active (Aligizaki et al., 2008; Cagide et al., 2009). Despite its high lethality in terrestrial animals, PLT has also been detected in various sh, crabs and sea anemona without causing any deleterious effects (Aligizaki et al., 2008). Studies on the bivalve mollusks are important from an ecological, economic and public health point of view (Malagoli et al., 2008) since those organisms are widely reported as the most contaminated seawater animals. For instance, PLT-like toxins can bioaccumulate in shellsh (Aligizaki et al., 2008) in fact they were found in mussels from the Caribbean Sea, Australia and recently from Greece

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(Gleibs and Mebs, 1999; Aligizaki et al., 2008). Animals may incorporate PLT by ltering and therefore entering the toxin in the food chain (Gleibs and Mebs, 1999). This fact results in a threat to human health and economic consequences for shellsh producers. However, little information is available on the effects that the bioconcentration of PLT in animal tissues may exert on bivalve mollusks (Malagoli et al., 2008) and particularly on mussels. The aim of this work was to study the toxicity of PLT in mussel cells. We have further compared its effects with other toxins common in European coast such as okadaic acid (OA) and azaspiracid-1 (AZA-1). 2. Materials and methods 2.1. Reagents and solutions for mussel cells isolation and assay AZA-1 was puried from mussels collected in Ireland during a harmful algal bloom in 2006 as described in Alfonso et al. (Alfonso et al., 2008). PLT from the soft coral Palythoa tuberculosa was purchased from Wako (Osaka, Japan). OA was from LC laboratories (Woburn, MA, USA). Alamar Blue was from Biosource International (Nivelles, Belgium). All other reagents were purchased either from SigmaAldrich or Panreac (Barcelona, Spain). Sea water (M): 0.5 ClNa, 0.01 ClK, 0.05 SO4Mg, 0.01 Cl2Ca, 0.005 HCO3Na. pH 7.8. Articial sea water (mEq/L): 512.5 Na, 10 K, 500 Cl, 7.5 OH, 20 mM HEPES, 1 mM EDTA. pH 7.0. Articial sea water with calcium: Variation of the articial sea water without EDTA and plus 5 mEq/L Ca2 and 10 mEq/L Cl. pH 7.0. 2.2. Mussel cells isolation Uncontaminated specimens of sea mussels Mytilus galloprovincialis of uniform size (810 cm in length) were purchased from commercial dealers in Galicia (North West coast of Spain). Cells from mantle and hepatopancreas were obtained as previously described (Vieytes et al., 1993). For each experiment, both hemi-mantles and hepatopancreas from approximately 20 mussels were excised, separately washed in ice-cold sea water and sliced with a bistoury in 1 mm fragments. Then, mantle and hepatopancreas portions were immersed in ice-cold sea water and gently agitated with a magnetic stirrer. After washing for 15 min the slices were rinsed twice with fresh ice-cold sea water and ltered through 200 mm nylon meshes three times. Finely chopped 510 g of mantle and hepatopancreas tissues were enzymatically dispersed with 0.7 IU/mL collagenase and 6 IU/mL dispase in 20 mL of articial sea water for 45 min at 37  C. The disaggregated tissue was ltered through a 200 mm nylon mesh. The treatment resulted in a suspension containing the mantle or the hepatopancreas cells, which were washed by centrifugation at 800 rpm for 5 min and re-suspended in articial sea water twice. The obtained cells were then re-suspended in articial sea water with calcium, and used when cellular viability determined by trypan blue exclusion test was !70%.

2.3. Metabolic activity assay Metabolic activity of mussel mantle or hepatopancreas cells was measured by using Alamar blue. This compound becomes uorescent when reduced, without affecting cell integrity (Ahmed et al., 1994). Therefore cell metabolic rate can be proportionally evaluated by measuring the uorescence intensity (Espina et al., 2009). Cells were seeded on Corning Costar 96-well plates (Schiphol-Rijk, The Netherlands) at a density of 100,000 cells per well. AZA-1 (100 nM and 1 mM), PLT (0.15, 0.75, 1.5, 7.5, 15 and 75 nM), or OA (0.5 and 1 mM) and vehicle (for the controls) were added to the cells. Finally, a 1:10 dilution of Alamar Blue was added and uorescence was measured by using a microplate uorescence reader FL600 (Bio-Tek/Vermont, U.S.A.). Cells were incubated in constant shaking at room temperature, and measurements were registered 3, 6 and 12 h after toxins addition. Results are presented as percentage of uorescence versus its respective control (cells without toxin); mean values S.E.M, with n ! 3. 3. Results Recently, it was reported the rst episode of shellsh contamination by PLT-like compounds from Ostreopsis species in Europe (Aligizaki et al., 2008). However, the cytotoxicity of PLT in mussels has not been investigated. We tested the effect of PLT on mussel cells by using Alamar Blue. We incubated the cells with increasing concentrations of this toxin ranging from 0.15 nM to 75 nM at different exposure times (Fig. 1). We found that from concentrations above 1.5 nM, PLT induced a great and progressive decrease in the metabolic rate of mussel mantle (Fig. 1a). The same trend was observed with hepatopancreatic cells (Fig. 1b). In fact, a dose-response decrease of metabolic activity was detected in response to PLT in both kinds of cells (Fig. 2). The IC50 calculated was 18.23 nM for mantle (Fig. 2a) and very similar, 16.17 nM for hepatopancreatic cells (Fig. 2b). Next, we compared the effect observed with other toxins that also appear on the European coasts. AZA-1 is a newly identied marine toxin reported to accumulate in mollusks from several northern European countries and documented to have caused severe human intoxications (Twiner et al., 2005). Several investigations have shown that AZA-1 is cytotoxic to a range of mammalian cell types (Vilarino et al., 2006, 2007, 2008a; Vale et al., 2007). We studied if AZA-1 was cytotoxic to mussel cells by applying the previous assays (Fig. 3). In this case, 100 nM, nor even 1 mM AZA-1 were able to induce any change in the metabolic range neither in mantle (Fig. 3a) nor in hepatopancreas cells (Fig. 3b) at any of the times registered. Then, we tested the cytotoxic effect of another marine toxin commonly found on the European Coasts: OA (Fig. 4). This toxin is a polyether fatty acid causing diarrheic shellsh poisoning (DSP) and is produced mainly by dinoagellates. Mussels are the main vectors of DSP (Svensson et al., 2003). Unlike PLT, we found that OA (0.5 and 1 mM) was unable to trigger any modication on the metabolic rate of mussel cells from the mantle (Fig. 4a) or from the hepatopancreas (Fig. 4b).

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Fig. 1. Effect of PLT on the metabolic rate of mussel cells. Mantle (a) or hepatopancreatic (b) cells were incubated for up to 12 h with 0.15, 0.75, 1.5, 7.5, 15 or 75 nM PLT and the metabolic rate was measured by monitoring the uorescence of Alamar Blue. Results are expressed as percentage of uorescence versus controls (100%) (Mean S.E.M.; n 4).

4. Discussion Given their feeding behavior, bivalves can accumulate molecules that are potentially dangerous for human health, including marine biotoxins (Ares et al., 2005). The fact that seafood products can be contaminated by several different classes of toxin, the increased complexity of toxin panels and the recent problem of emerging toxicities, probably related to the global climatic change, should encourage us to study the effect of toxins in seafood. PLT is considered as one of the most toxic molecules occurring in nature, and it exerts a wide spectrum of pharmacological effects in vitro and in vivo such as cellular cytoskeleton disruption and skin tumors promotion in mice (Ares et al., 2005; Louzao et al., 2007, 2008; Wattenberg, 2007). As other phycotoxins, PLT are taken by lterfeeders and can be accumulated in animal tissues. Toxins

pass over the food chain and nally may provoke severe, or even lethal, intoxications to humans (Taniyama et al., 2003; Malagoli et al., 2008). PLT compounds appear to be present in edible sh for instance in Hawaiian reef species, both carnivorous and herbivorous. It was surprising to nd sh species with high PLT activity also in their esh, given the water soluble nature of PLT (FAO, 2004). In addition to sh, PLT has caused human poisonings due to the consumption of several species of xanthid crabs from the Philippines (Yasumoto et al., 1986). In bivalves, PLT activity was found in clams and mussels in Europe (Aligizaki et al., 2008) but no morphological or physiological changes were reported in those animals. PLT compounds attack all mammalian cells that have been studied (Wu, 2009). However the interaction of these toxins with cells from sh, crabs or bivalves has been quite unexplored.

Fig. 2. Dose-response effect of PLT in the metabolic rate of mussel mantle and hepatopancreatic cells after 6 h of incubation. Results are presented as percentage of Alamar Blue uorescence relative to control cells (100%); mean values s.e.m, with n 4.

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Fig. 3. Effect of AZA-1 on the metabolic rate of mussel cells. Mantle (a) or hepatopancreatic (b) cells were incubated for up to 12 h with 100 nM or 1 mM AZA-1 and the metabolic rate was measured by monitoring the uorescence of Alamar Blue. Results are expressed as percentage of uorescence versus controls (100%) (Mean S.E.M.; n 3). Mantle cells incubated with 1 mM AZA-1, n 2.

In this work we tested if PLT and other toxins such as OA and AZA-1 modify the metabolic activity of mussel cells by using Alamar Blue. This is a non-toxic uorescent dye, which allows a dynamic cytotoxic detection. OA does not inuence viability either in mantle or in hepatopancreas mussel cells, while this toxin signicantly decreases metabolic rate and modify cytoskeleton in human neuroblastoma cells (Vilarino et al., 2008b; Espina et al., 2010). As OA, AZA-1 does not reduce the viability of mussel cells. However, PLT dramatically decreases the percentage of mantle or hepatopancreas live cells. Therefore our results indicate that the cytotoxic effect reported to occur in neuroblastoma and hepatocytes can also be induced by the toxin in mussel cells (Cagide et al., 2009; Espina et al., 2009; Ledreux et al., 2009). Previously, we found that 2 g homogenate of mussels naturally contaminated with PLT in Greek coasts contained approximately 200 ng PLT equivalents (Espina et al., 2009). In this work, quantities of toxin 2 times smaller than those we found in greek mussels already induced a fall in cell viability. Taking both data into account, mussels can accumulate enough quantities of PLT to damage their mantle or hepatopancreas cells. Therefore,

PLT affects bivalve cells, despite accumulating this toxin, indicating that the harmlessness of PLTs for mussels might be only apparent (Gleibs and Mebs, 1999). Such data could be of fundamental importance in evaluating the real ecological impact of the toxin and may contribute to clarify how highly toxic molecules such as PLT can be concentrated in some bivalves without any apparent external effects. In previous studies mussel exhibit different behavior and cellular responses when exposed to other toxins such as saxitoxin (STX). In this case bivalves are described as STX non-sensitive species. Effectively, the whole animal is resistant and bioaccumulate the toxin but also the toxin has no effect on the mussel cells (Botana et al., 1992; Louzao et al., 1992a). It is also possible that mussels change their behavior and ability to concentrate the toxin depending on the relative toxicity of the compounds, the history of harmful algal bloom in the ecosystem or some modulatory activity of the cells as was previously described in crabs exposed to saxitoxin (Louzao et al., 1990, 1992b). In mammalians the cell surface is the primary site of action of PLT and the disruption or alteration of transmembrane ionic gradients modify signal transduction. In

Fig. 4. Effect of OA on the metabolic rate of mussel cells. Mantle (a) or hepatopancreatic (b) cells were incubated for 6h with 0.5 or 1 mM OA and the metabolic rate was measured by registering the uorescence of Alamar Blue. Results are expressed as percentage of uorescence versus controls (100%) (Mean S.E.M.; n 3).

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mussels it has been recently discovered that PLT plays an active role in M. galloprovincialis immunocyte signaling transduction pathways, increasing the phagocytic activity and promoting the involvement of p38 mitogen-activated protein (MAP) kinase in phagocytosis (Malagoli et al., 2008). Nevertheless, PLT could have other important effects in mussels. The receptor for PLT is the cytoplasmatic membrane Na/K ATPase. The sodium pump is converted by the toxin in a non-specic ion channel (Hilgemann, 2003; Takeuchi et al., 2008). This fact results in a membrane depolarization due to K efux and Na inux to cells (Louzao et al., 2006; Wang, 2008). Subsequently, the membrane depolarization may open voltage dependent Ca2 channels while Na inux may load cells with Na and favors Ca2 uptake by the Na/Ca2 exchanger (Shimahara and Molgo, 1990; Louzao et al., 2007; Cagide et al., 2009). The ionic disorder induced by PLT triggers a series of cellular events that lead to its cytotoxic effect in mammalian cells. Our data also indicate that PLT exhibit toxicity in mussel cells suggesting a toxin activity related to Na/K ATPase as well as in mammalian cells. This could be engaged with the fact that the Na/K ATPase is a key enzyme involved in marine animals osmoregulation and salinity adaptation. However, we did not detect changes in mussel cells metabolic rate when they were exposed to other phycotoxins usually found in mussels. On the base of the present study it is clear that PLT seriously perturbs mussel cells therefore cells are not adapted to those toxins being very sensitive to them. Taking into account that mussel cells are sensitive to the toxin we could expect that PLT is harmful to mussel. Opposite the mollusks enable PLT sequestration and accumulation with lack of apparent modications. The mechanism of masking or inhibiting the toxins activity while it is transferred and stored in the body of mussel is still unknown as it happens with many other marine organisms (Gleibs and Mebs, 1999). Like other groups of phycotoxins, such as saxitoxins, pectenotoxins or brevetoxins, PLTs and its related compounds could be metabolized by shellsh to form more or less toxic analogues (Plakas et al., 2002, 2004; Artigas et al., 2007) However we could speculate that there is an absence of PLT metabolization and detoxication in M. galloprovincialis due to this mussel is not adapted to the presence of PLT on the European coasts.

Conict of interest None. References

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Acknowledgements This work was funded with the following grants: From Ministerio de Ciencia y Tecnologa, Spain: AGL2006-08439/ALI, AGL2007-60946/ALI From Xunta de Galicia, Spain: GRC 30/2006, and PGIDT07CSA012261PR, PGDIT 07MMA006261PR, 2008/ CP389 (EPITOX, Consellera de Innovacion e Industria, programa IN.CI.TE.), 09MMA003261PR. From EU VIth Frame Program: IP FOOD-CT-2004-06988 (BIOCOP), STREP FOOD-CT-2004-514055 (DETECTOX) and CRP 030270-2 (SPIES-DETOX) From EU VIIth Frame Program: 211326 CP (CONffIDENCE); STC-CP2008-1-555612 (Atlantox)

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