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Introduction

In the Galician Rias, Dinophysis acuminata and Dinophysis acuta are the main species of toxic microalgae, and have been shown to produce okadaic acid and dinophysistoxin-2 (Fernández et al. 1998). Okadaic acid, diniphysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) have been identified in most Diarrhetic Shellfish Poisoning (DSP) episodes in Galician mussels (Gago-Martinez et al. 1996), as well as being found in Irish (Carmody et al. 1996) and Portuguese (Vale and Sampayo 2002) shellfish.

OA and Dinophysistoxins (DTX’s) are the principal toxic compounds causing DSP in humans (Yasumoto et al. 1985). These compounds are lipophilic polyether molecules produced by dinoflagellates Dinophysis sp. and Prorocentrum sp. genera (Yasumoto et al. 1978; Murata et al. 1982).

Mussels, like other bivalves, can accumulate DSP toxins during algal blooms of these microalgal species. Accumulation takes place mainly in the digestive gland (Blanco et al. 2007) and causes a negative economic impact to the shellfishing industry in the Galician rias on an annual basis. Following ingestion of contaminated mussels by humans the DSP toxins can cause a gastrointestinal disease and result in a serious public health problem.

The genetic selection of molluscs with a greater capacity for detoxification, in order to reduce depuration times, could help to reduce the resultant significant economic and health problems.

There are few studies concerning the metabolism of biotoxins in bivalves, although some authors have suggested involvement of detoxification enzymes (Kodama and Sato 2002; FRS Marine Laboratory 2004) and described the induction of some antioxidant enzymes in crustaceans and scallops in the presence of toxic dinoflagellates (Campa-Córdova et al. 2009).

This work is a preliminary study of the metabolism of okadaic acid in Mytilus galloprovincialis. We analysed a diverse range of enzymes potentially involved in xenobiotic metabolism and follow enzyme activity variation in relation to OA accumulation during three different toxic blooms of D. acuminata and D. acuta in the Ria de Vigo.

Material and Methods

Samples

Mussels (Mytillus galloprovincialis) were sampled fortnightly from floating rafts of the Vigo estuary (NW Spain) from June 2001 until August 2002. For each sampling, 80 adult individuals of 6–8 cm in length were randomly collected. The digestive glands of 30 mussels were dissected, immediately frozen in liquid nitrogen, pooled and stored at −80 °C until the enzymatic assays. A section of mantle tissue was also dissected, fixed in Bouin’s solution and histologically processed using routine histological techniques (paraffin embedded, 5 μm sectioning, stained with Harris’ haematoxylin-eosin solution and analysed microscopically) to determine the sex of each mussel.

The soft tissues of the other subsample (50 mussels) were pooled, homogenized, lyophilized and stored for drying until toxin analysis. In Mytilus, enzymatic activity varies with the reproductive status, sex and environmental parameters (Borkovic et al. 2005; Bochetti and Regoli 2006; Monserrat et al. 2007; Verlecar et al. 2008; Cravo et al. 2009). In this study, the histological analysis showed that most of the mussels collected on the same date were at the same gametogenic stage. Because of this and because we separated males and females following collection, we felt justified in pooling each sample from the same sampling location.

Enzymatic Analysis

Following sex determination the digestive glands were pooled for each sampling date and by sex and then homogenized in 20 mM Tris-HCl buffer pH7.6 (1:4, w:v) containing 0.5 M sucrose, 0.15 M potassium chloride (KCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM â-mercaptoethanol and 0.1 mM of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 4 °C at 500 × g for 30 min and the resulting supernatant further centrifuged at 12,000 × g for 45 min. This last supernatant was considered to be the post-mitochondrial fraction, on which all enzymatic activities were assayed by spectrophotometric methods at 25 °C. Assays were performed in triplicate for each sample.

Cytochrome P450 reductase (CPR) was determined as described by Livingstone and Farrar (1984) using NADPH and NADH as electron donor and cytochrome c as substrate. 7-ethoxi-resorufine-O-deetilase (EROD) was assayed by the method of Burke and Mayer (1974). Xanthine oxidoreductase (XDH) was determinated following the method described by Lallier and Walsh (1991). DT-diaphorase activity (DTD) was determined as described by Ernster (1967) and Livingstone et al. (1992). Glutathione-S-transferase (GST) was determined as the conjugation enzyme by the method of Habig and Jakoby (1981) Among antioxidant enzymes, superoxide dismutase (SOD), total and selenium-dependent glutathione peroxidase (GPx-tot; GPX-Se); glyoxalases I and II (GLO I, GLO II) and Catalase (CAT) were assayed respectively, by the methods described by Ewing and Janero (1995), Lawrence and Burk (1976), Principato et al. (1983) and Aebi (1984). Glutathione reductase (GR) was assayed by the Ramos-Martínez et al. method (1983), as an enzyme involved in the redox cycle of glutathione, which yields an adequate concentration of reduced glutathione for GPX and GLO I activities. Activities were expressed in UI or mUI per gram of tissue.

Extraction and Analysis of Toxins

Toxin extraction for each sample date was carried out with 0.4 g of lyophilized mussel (equivalent to 2 g of wet weight). Three extractions were carried out adding 80 % MeOH at a ratio of 1:4 (weight: volume) according to Quilliam 1995. Extracts were clarified by centrifugation (10,000 × g for 10 min at 20 °C) and transferred into a volumetric flask through syringe filters of 0.22 μm. The extracts were evaporated in a rotavapor, resuspended freshly in MeOH 80 %, and then filtered through ultrafree centrifugal filters of 0.45 μm.

Aliquots of each extract were hydrolysed at 75 °C for 40 min. with 2.5 M NaOH, and the reaction stopped by adding 2.5 M HCl.

The presence of okadaic acid in the samples was determined by HPLC–MS/MS with a Surveyor MS HPLC system, coupled to a Deca XP plus ion trap mass spectrometer (Thermo Fisher Scientific) with an electrospray interface, following the method of Gerssen et al. (2009). The chromatographic separation was carried out using a Gemini NX C18 column. The mobile phase consisted of 100 % water (A) and acetonitrile: water (95:5 %) (B), both containing 2 mM ammonium formate and 50 mM formic acid (pH 2.6). An isocratic elution consisting of a 10 % mobile phase B and 90 % mobile phase A, was run. The mobile phase flow was 0.4 mL/min and the injection volume was 20 μL.

OA was quantified by comparison with reference materials from NRC-CRM, Canada.

Results and Discussion

During our study three toxic episodes, caused by D. acuminata and D. acuta, occurred between June 18, 2001 and August 6, 2002 (INTECMAR: www.intecmar.org), during which our results showed that OA was accumulated by mussels (Fig. 19.1). DTXs were not detected in any samples.

Fig. 19.1
figure 1

OA accumulation and variation of CPR, GST, GR, GPX, CAT and GLO I activities from M. galloprovincialis digestive gland during three toxic episodes of D. acuminata. Bars: concentration of OA accumulated in mussel soft tissues. Enzymatic activities in females and in males (●)

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Considering all enzymatic activities evaluated, only CPR, GST, GR, GLO I, GPXtot and CAT showed significant variations, which were related to the accumulation of OA in mussels.

The CPR activity showed a significant increase, mainly in males, inversely related to decrease of OA accumulated (r: 0.375, p < 0.05) (Fig. 19.1a; Table 19.1). This enzyme provides electrons to different oxygenases from the endoplasmic reticulum, between them to the oxygenase- dependant cytochrome P450, which is involved in xenobiotic metabolism, steroids and lipid signalling synthesis, sterol synthesis or the metabolism of desaturation or elongation of fatty acids, establishing a microsomal electron transport sequence known as “mixed function oxidase system”. The, oxidation-reduction reactions of this system allow hydroxylation of multiple molecules and converts them into polar molecules in order to facilitate their elimination (Guengerich 1988). The relationship between CPR and OA in mussels is consistent with the involvement of some microsomal monooxygenases in the metabolism of OA. Other authors have described similar results in hepatocyte cultures from vertebrates (Tamaki et al. 2005; Guo et al. 2010).

Table 19.1 Correlation factors between diverse enzyme activities related to xenobiotic metabolism from mussel digestive gland and accumulation of OA
Full size table

The metabolites produced by the mixed function oxidase system are not easily eliminated and are usually more toxic than the initial compounds. Such metabolites can be conjugated with endogenous reduced glutathione to increase its polarity and hence make it easier to eliminate them. GST catalyzes the conjugation of a variety of endogenous and xenobiotic substrates with reduced glutathione (GSH) (Mannervik 1985; Listowsky et al. 1988). GST has an important role in preventing peroxidation and detoxification of toxic substances. In this work we observed an increase of this activity, parallel to decrease of OA and which was significantly correlated with CPR activity (r: 0.516, 0.544 in males and females, respectively; p < 0.01) (Fig. 19.1b; Table 19.1), suggesting its possible participation in OA metabolism.

The GST activity depends on the presence of reduced glutathione. The redox balance of cellular glutathione is maintained by glutathione reductase (GR). This enzyme catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) (Ulusu and Tandogan 2007), which is also necessary for other enzymatic activities, such as glutathione peroxidase and glyoxalase I. In our study, GR activity showed a different behaviour in relation to OA accumulation, which was dependant on the sex of the mussel. In males we observed an increase of GR activity at the beginning of intoxication and then a decrease parallel to GST activity, when the OA began decreasing. Thus our results show a significant correlation between GR and GST activity (r: 0.498, p < 0.01) and between GR and OA accumulated (r: 0.483, p < 0.01). In female mussels there are no notable differences with males, although our results shows a GR activity inhibition at the beginning of the toxic episodes (Fig. 19.1c), which is negatively correlated with OA accumulation (r: −0.514, p < 0.01) (Table 19.1).

The oxidative reactions catalyzed by the mixed function oxidase system generate large amounts of reactive species of O2 and other molecules, such as α oxaldeydes, which are highly toxic. Such molecules can be eliminated by the activity of other enzymes concerned with oxidative defense. Some authors have also described the induction by OA of these enzymes in vertebrate cell cultures (Fujii et al. 1994; Matias et al. 1999) and mussels (Auriemma and Battistella 2004).

Among the other enzymes of oxidative defense, the glioxilases I and II catalyze the coordinated detoxification of reactive α oxaldeydes with mutagenic and cytotoxic activity, converting them into their corresponding α -hydroxy acids (thiol ester intermediaries) (Regoli et al. 1996).

The glioxalase I is also a GSH-dependent enzyme. α oxaldeydes and glutathione form spontaneously an intermediary hemithioaceatal, which is transformed to S-D-lactoilglutathione by the GLO I activity. This S-D-lactoilglutathione is subsequently hydrolyzed to d-lactate and glutathione (GSH) by GLO II enzyme. Our results show an increase of GLO I activity with OA intoxication in both sexes (Fig. 19.1d) with a statistically significant correlation in males (r: 0.370, p < 0.05) (Table 19.1). This enzyme also shows a significant correlation with GR (r: 0.771, p < 0.01) and GST (r: 0.737, p < 0.01) activities in males, and with CPR (r: 0.334, p < 0.05) and GR (r: 0.557, p < 0.01) activities in females (Table 19.1). These results are consistent with induction by OA of GLO I in Mytilus as also obtained by Auriemma and Battistella (2004).

Other oxidative defence enzymes that seem to be related to the episodes of intoxication by OA in mussels are glutathione peroxidase (GPXtot) and catalase (CAT). GPXtot activity increases in both sexes during intoxication, showing in females a negative correlation with accumulated OA (r: −0.366, p < 0.01) (Fig. 19.1e; Table 19.1) and positive one with CPR (r: 0.440, p < 0.01), GST (r: 0.608, p < 0.01) and with GLO I (r: 0.312, p < 0.0) activities. In males its activity is only correlated with GST (r: 0.430, p < 0.01) and GLO I (r: 0.412, p < 0.01) (Table 19.1). On the other hand, CAT activity has no correlation with accumulated OA (Fig. 19.1f), but shows significant correlation with CPR (r: 0.338, p < 0.0 5) and GST (r: 0.416, p < 0.01) in males and with GPXtot (r: 0.317, p < 0.05) in females.

Despite the preliminary nature of this work, the results obtained suggest the involvement of the microsomal monooxygenase enzymatic system dependent on cytochrome P450 in the okadaic acid biotransformation in Mytilus galloprovincialis. Moreover, the different enzymatic correlations in males and females seem to indicate sexual differences in the metabolic pathways followed. However, to confirm this and to define other possible enzymes and pathways involved in OA degradation and elimination in mussels further studies will be required.