Aquatic Toxicology 157 (2014) 94–100

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Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): Regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress Concetta Maria Messina a,∗,1 , Caterina Faggio b,1 , Vincenzo Alessandro Laudicella c , Marilena Sanfilippo b , Francesca Trischitta b , Andrea Santulli a,c a University of Palermo, Department of Earth and Marine Science DiSTeM, Marine Biochemistry and Ecotoxicology Laboratory, Via G. Barlotta 4, 91100 Trapani, Italy b Department of Biological and Environmental Science, University of Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy c Consorzio Universitario della Provincia di Trapani, Istituto di Biologia marina, Via G. Barlotta 4, 91100 Trapani, Italy

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Article history: Received 20 May 2014 Received in revised form 11 August 2014 Accepted 3 October 2014 Available online 13 October 2014 Key-words: Mytilus galloprovincialis SDS (sodium dodecyl dulfate) Cell volume regulation Biomarkers of oxidative stress Endogenous antioxidants

a b s t r a c t In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1 mg/l to 1 mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (p < 0.05). Consequently, two enzymes involved in ROS scavenging, superoxide dismutase (SOD) and catalase (CAT) have higher activities and the proportion of oxidized glutathione (GSSG) is higher in hepatopancreas and mantle of treated animals, compared to untreated animals (p < 0.05). Furthermore Hsp70 demonstrates an up-regulation in all the analyzed tissues of exposed animals, attesting the stress status induced by the surfactant with respect to the unexposed animals. The results highlight that SDS, under the tested concentrations, exerts a toxic effect in mussels in which the disruption of the osmotic balance follows the induction of oxidative stress. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nowadays the uses of detergents are important parts of anthropogenic activities and since the second half of the twentieth century there has been an exponential growth in their consumption (Singh et al., 2007). This situation causes a progressively increasing discharge of large amount of these products which, via sewage, reach the aquatic environment, affecting the organisms living there. Thus there is the need for adequate biomarkers of exposure (Pettersson et al., 2000; Nunes et al., 2008).

∗ Corresponding author at: Laboratorio di Biochimica Marina ed Ecotossicologia (LaBMEco), Via Barlotta 4, Trapani 91100, Italy. Tel.: +39 0923560162; fax: +39 09160666. E-mail address: [email protected] (C.M. Messina). 1 These two authors contributed equally to the work. http://dx.doi.org/10.1016/j.aquatox.2014.10.001 0166-445X/© 2014 Elsevier B.V. All rights reserved.

Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant (Surface Active Agent, SAA), common in personal care and household products, characterized by high amphiphilicity and adsorption ability. Toxicity of SDS has been demonstrated upon bacteria, microalgae, crustaceans, echinoderms and fish (Mariani et al., 2006). The basis of its toxicity seems to be mainly related to the alteration of the cellular ionic balance caused by cellular membrane permeability alterations (Grant and Acosta, 1996) and to the induction of oxidative stress (Bromberg and Pick, 1985) which, in turn, can generate other physiological and biochemical stresses. Mussels, are commonly used as sentinel organisms in biomonitoring studies due to their wide geographical distribution and ability to accumulate contaminants (Viarengo et al., 2007). These organisms, being osmo-conformers, represents a relevant model for studies of physiological regulatory responses, as they can resist to large fluctuations of environmental parameters, by triggering efficient adaptive mechanisms (Torre et al., 2013a). Among the

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mechanisms of adaptation, Regulatory Volume Decrease (RVD), constitutes an integrative process that most animal cells, also in mussels, perform to counteract hypo-osmotic swelling. The maintenance of cell volume and its restoration after the “disturbance” following the exposure to xenobiotics, allows cells to protect themselves from the lysis and, at the same time, to fulfill their overall role in the organism (Torre et al., 2013b). The disruption of membrane potential by anionic SAAs has been shown to alter the osmoregulation ability of mussels (López-Galindo et al., 2010a) and fish (Barbieri et al., 2002; López-Galindo et al., 2010b). Nowadays, the massive discharge of anionic surfactants has increased the awareness on the effects of these compounds on antioxidant system of bivalves and fish (Alvarez-Munoz et al., 2009; da Silva and Meirelles, 2004; Guilhermino et al., 2000; Nunes et al., 2008; Ostroumov, 2003; Romanelli et al., 2004; Wu et al., 2005; Zhang et al., 2005). The oxidative stress, which is mostly caused by an over-production of reactive oxygen species (ROS) with respect to the endogenous antioxidant potential, has been largely used in the assessment of effects induced by chemicals and contaminants on marine organisms (Livingstone et al., 2000), and hence also for SDS exposure (Jifa et al., 2005; Liu et al., 2010; Nunes et al., 2008). However, the molecular mechanisms of toxicity of SDS are not well understood. The aim of this study is to evaluate the effects of SDS in the Mediterranean mussel (M. galloprovincialis), exposed for 18 days under laboratory conditions, using an environmentally relevant concentration range already tested by others (Jifa et al., 2005; Liu et al., 2010; Lürling, 2006; Nunes et al., 2008; Olkowska et al., 2011; Pettersson et al., 2000). Among the physiological parameters of stress we evaluated RVD which earlier was shown to be an interesting and sensitive marker of exposure to contaminants in mussels (Torre et al., 2013a). As this biomarker is a fast, easy and reliable indicator of xenobiotic effects in mussels (Torre et al., 2013a), we decided to monitor it not only at the end, but also in the middle of the experiments (10 days). To evaluate the oxidative stress caused by the exposure of mussels to SDS, we analyzed the production of ROS, and screened the activities of two antioxidant enzymes, SOD (Superoxide Dismutase) and CAT (Catalase) together with the three-amino acid peptide glutathione (GSH), as main antioxidant defenses in tissues and organisms. Last, as general marker of stress and consecutive adaptation, we evaluate the levels of Hsp70 (Heat Shock Protein). Its expression change have been demonstrated to occur in response to a wide variety of chemical and physical inputs, as well as to conditions causing oxidative stress conditions (Livingstone, 2003; Dimitriadis et al., 2012).

2. Materials and methods 2.1. Animal collection Mediterranean mussels, M. galloprovincialis (7–8 cm shell length), were collected in Faro, Messina (N/E Sicily, Italy), transferred in 10 min to a tank containing synthetic natural sea water (SNSW, Nutri-SeaWater® Aquarium Saltwater, pH: 8 ± 0.1; salinity: 36 ± 1 ppt; temperature: 17 ± 1 ◦ C) with continuous aeration. After 3 days of acclimation, mussels were randomly divided into 3 tanks (16 liter) with 35 animals each, filled with the same SNSW and kept under the conditions described above. Each tank was subjected to a different concentration of SDS: CO (control); 0.1 (containing 0.1 mg/l SDS); 1 (containing 1 mg/l SDS). The exposure lasted for 18 days. SDS (purity >99%) was obtained from Sigma–Aldrich Chemical Co (St. Louis, MO, USA). The mussels were fed once a day with algal slurry (Liquifry marine, Interpet, Dorking, England), SNSW was changed weekly and environmental parameters were daily monitored with a multiparametric probe YSI 85 System (YSI

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Incorporated USA) and with a pH-meter HANNA HI 83140. After 18 days of continuous exposure, the organisms were sampled for biochemical analyses. 2.2. Isolation of digestive cells and RVD experiments The RVD experiment was done at 10 days and at the end of the experiment (18 days). For both experiments, 2 pools, composed by the digestive glands of 5 animals were isolated according to the method of Dailianis and Kaloyianni (2007) in order to obtain an adequate number of cells. Digestive glands were cut in pieces and washed with a Ca2+ and Mg2+ free solution (Sol. 1: NaCl 600 mM; KCl 12.5 mM; HEPES 20 mM), transferred to a test tube containing 0.01% collagenase (type IV-activity P125 CDU/mg; CDU = collagenase digestion units, Sigma–Aldrich, St. Louis, MO, USA) dissolved in Sol. 1. The test tube was gently stirred for 60 min at 18 ◦ C in a thermostatted bath. Afterwards the suspension was filtered through 200 ␮m and 75 ␮m nylon filters. The cells were suspended in physiological saline solution (Sol. 2: NaCl 550 mM; KCl 12.5 mM; MgSO4 8 mM; CaCl2 4 mM; glucose 10 mM, HEPES 20 mM), washed twice by centrifugation (500 rpm/10 min/4 ◦ C) and then re-suspended in sol. 2. Before the experiments the cells were maintained in physiological saline solution (Sol. 2) at 18 ◦ C for 1 h to re-establish ionic concentration on either side of the cell membrane. Before the RVD experiments, the viability of the cells and the quality of their lysosomes was evaluated with Trypan blue exclusion method and neutral red retention assay (NR) (which measures the stability of lysosomal membrane). For NR the dye retention was evaluated after an incubation period of 30 and 60 min, according to Repetto et al. (2008). Isolated digestive cells did not exhibit differences in viability and lysosomal stability between exposed and control animals. For RVD experiment the isolated cells were visualized and measured by the method described in a previous paper (Torre et al., 2013b). One drop of cell suspension was placed on a glass slide pretreated with poly-lysine to facilitate cell adhesion. Two thin strips of double-sided adhesive were placed at the upper and lower edges of the glass slide to support the cover slip and to create an interspace in which the hypotonic experimental solution was then added (Sol. 3: NaCl 350 mM; KCl 12.5 mM; MgSO4 8 mM;CaCl2 4 mM; glucose 10 mM, HEPES 20 mM). Cells were placed at one side of the cover slip with a pipette and were absorbed at the opposite side with strips of filter papers. This allowed a rapid change (in a few seconds) of the solution in the interspace. Cells were observed with a light microscope (Leitz Diaplan) and video-metric measurements were carried out on digestive cells. Cell images were digitized using a color video camera (Sony) connected to a PC. Individual cells were selected; images were taken at various time intervals and recorded on PC. The profile of the cells was drawn with the aid of ImageJ (NIH, Maryland, USA). The cell areas for each experimental condition (Aexp ) were compared to the areas measured in isotonic solution (Ai ) at the beginning of the experiment. Consequently the data are reported as relative area Aexp /Ai . 2.3. Tissue dissection and total protein extraction At the end of the 18 days, both control and exposed mussels were sampled. Gill (G), Hepatopancreas (H) and Mantle (M) tissues were dissected and cold-homogenized in mussel physiological buffer (MPB) (1:10, w/v) for protein extraction (Livingstone et al., 1992). For each treatment, tissues were recovered from five organisms and each extraction was performed in duplicate. The total protein content of each sample was determined according to Lowry et al. (1951).

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2.4. Evaluation of intracellular ROS Intracellular ROS were analyzed by the dichlorodihydrofluorescein-diacetate (DCF-DA) method as reported by Kang et al. (2005). DCF-DA is oxidized to dichlorodihydrofluorescein (DCF) by ROS. Each sample in MPB (100 ␮l) was exposed to 10 ␮l of DCF-DA in HBSS (5 ␮g/ml), incubated for 5 min at 37 ◦ C to allow the oxidation of the DCF-DA and the fluorescence successively read on a spectrofluorometer (excitation wavelength 485 nm, emission 530 nm; Varian Cary Eclipse). The results are expressed as relative fluorescence/␮g of total proteins (rf/␮g T.P.).

analysis of the variance (ANOVA). To assess the differences of RVD, between control and treatments at 18 days, a two-way ANOVA was employed. The statistical differences for the oxidative stress markers (ROS, SOD, CAT and GSSG), for each tissue, were analyzed by a one-way ANOVA. The differences among the mean values were subjected to Student–Newman–Keulls test (SNK). Prior to analysis, the degree of heterogeneity was assessed by the Cochran test (Underwood, 1997). Differences were considered significant at p < 0.05. The analyses were performed using the program STATISTICA (v. 8.0, Statsoft Inc., USA). 3. Results

2.5. Evaluation of scavenger enzyme activities and glutathione level SOD activity was measured by the reduction of xanthine oxidase to uric acid and H2 O2 which reduces NBT to NBT-formazan (Wang et al., 2010). To the tissue samples homogenized in MPB an equal volume of assay buffer was added (0.05 M Na2 CO3 pH 10.1; 3 mM xanthine; 0.75 mM NBT; 3 mM EDTA; 1.5 mg/ml BSA). The reaction was started by the addition of 50 ␮l of xanthine buffer (Xanthine oxidase in H2 O, 0.1 mg/ml), the samples were then incubated for 30 min at room temperature. Afterwards the reaction was stopped by the addition of 500 ␮l of copper chloride (6 mM) followed by centrifugation at 1500 × g for 10 min. The absorbance of supernatant was analyzed by spectrophotometer (Varian Cary 50 Scan) at 480 nm. Results were expressed as unit of SOD per microgram of total protein (U/␮g T.P.) and reported in relation to each respective control. CAT activity was measured according to Kang et al. (2005). To 50 ␮l of homogenates in MPB, 200 ␮l of 50 mM of Phosphate Buffer (pH 7) and 12 ␮l of H2 O2 were added, followed by an incubation at 37 ◦ C for two min. The samples were then analyzed by the spectrophotometer (Varian Cary 50 Scan) over 5 min at 240 nm. The change in absorbance was the rate of H2 O2 breakdown by CAT which was expressed as a unit of CAT per microgram of total protein (U/␮g T.P.) and reported in relation to each respective control. For glutathione determination, MPB lysates were assayed using a GSH/GSSG colorimetric assay kit (Calbiochem-Novabiochem Corporation, San Diego, CA, U.S.A.) according to the manufacturer’s protocol. 2.6. Immunoblot detection of Hsp70 Levels of Hsp70 were evaluated by immunoblotting. Equivalent amounts of proteins (40 ␮g) were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane using a Trans Blot Turbo Transfer System (Bio-Rad). The correct total protein loading was confirmed by red ponceau staining. The filters were then used for detection with primary monoclonal anti-Hsp70 antibodies (Sigma-Aldrich Chemical Co, St. Louis, MO, USA) and the appropriate anti-mouse horseradish peroxidase-conjugated secondary antibodies (Tebu-Bio). Immuno-reactive signals were detected using enhanced chemo-luminescent (ECL) reagents (Bio-Rad). Images were obtained, visualized, photographed and digitalized with Chemi Doc XRS (Bio-Rad). The images were then analyzed with Image Lab software (Bio-Rad). The results were expressed as fold increase of the band of each treatment in relation to the respective control and represent the mean value of at least three separate experiments. 2.7. Statistical analysis The data are given as mean ± SEM. The statistical differences among the three treatments (CO, 0.1 and 1) were evaluated by the

3.1. RVD experiments in digestive cells In order to evaluate the response to hypotonic solution, isolated digestive cells from animals grown in CO were exposed to a rapid change of osmolarity (1100–800 mOsm/kg). As shown in Fig. 1, the cells exposed to the rapid change of osmolarity, initially increased their size and then tended to return to their initial volume. It was observed that the cells reached their maximum swelling, corresponding to an increasing of 15% of volume, after 1 min of exposure to the hypotonic medium. Thereafter, they exhibited an RVD response. In contrast, digestive cells, isolated from animals exposed to SDS (0.1 mg/l and 1 mg/l) for 18 days, were not able to perform RVD (Fig. 1). The results were comparable to those obtained at 10 days (results not shown). 3.2. ROS Production The results related to ROS production are shown in Fig. 2. SDS exposure caused an overall increase of ROS in all analyzed tissues with respect to each control. In gill tissue the maximum ROS level was found in the 0.1 mg/l SDS treatment (1.7 ± 0.15 rf/␮g T.P.) followed by the 1 mg/l one (1.33 ± 0.04 rf/␮g T.P.), both higher than CO− (0.7 ± 0.2 rf/␮g T.P.) (p < 0.01) (Fig. 2). Similarly, hepatopancreas samples demonstrated a significant increase of ROS level from 0.4 ± 0.04 rf/␮g T.P. in the CO− , to 1.33 ± 0.21 rf/␮g T.P. and 1.57 ± 0.11 rf/␮g T.P. in lots treated with 0.1 and 1 mg/l SDS (p < 0.05) (Fig. 2). Mantle, likewise, showed a clear oxidative stress status as demonstrated by the significantly increased level of ROS in treated samples with respect to the CO (p < 0.05). The level increased with increasing SDS concentration (Fig. 2). 3.3. Scavenger activity The analysis of SOD activity demonstrated an increase (p < 0.05) of the enzyme activity in mantle and hepatopancreas as can be

Fig. 1. Relative area changes of the isolated digestive gland cells of M. galloprovincialis exposed to hypotonic solution, after 18 days of exposure to SDS (0.1–1 mg/l); number of cells = 40; The values are mean ± SEM; Control (); 0.1 mg/l (); 1 mg/l (䊉).

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Fig. 2. ROS production in M. galloprovincialis tissues (gills, G; hepatopancreas, H; mantle, M) after 18 days of exposure to SDS (0.1–1 mg/l). Data are expressed as relative fluorescence/␮g of total proteins (RF/␮g T.P. ± SEM). * p < 0.05; ** p < 0.01.

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Fig. 5. Relative content of GSSG in M. galloprovincialis analyzed tissues (gills, G; hepatopancreas, H; mantle, M) after 18 days of exposure to SDS (0.1–1 mg/l). Data are expressed as GSSG levels relative to each control (value = 1) ± SEM; *p < 0.05; **p < 0.01.

Fig. 3. SOD activity in M. galloprovincialis tissues (gills, G; hepatopancreas, H; mantle, M) after 18 days of exposure to SDS (0.1–1 mg/l). Data are expressed as unit for ␮g of total proteins (Unit/␮g T.P. ± SEM) and compared to each control. *p < 0.05; **p < 0.01.

observed in Fig. 3. The results are expressed as enzyme activity for ␮g of T.P. with respect of the control for each tissue. Results suggest that SOD activity was not increased by SDS treatment in gills (Fig. 3). The increased activity of SOD in hepatopancreas and mantle of M. galloprovincialis exposed to SDS was associated with a subsequent stimulation of CAT activity (Fig. 4). Similar to SOD activity, SDS treatment in gills did not affect CAT activity (Fig. 4). The organisms are experiencing oxidative stress, since the princi-

Fig. 6. Levels of Hsp70 in M. galloprovincialis tissues after 18 days of exposure to SDS (0.1–1 mg/l). (A): gills, G; (B): hepatopancreas, H; (C): mantle, M. The intensity of the bands was determined by densitometry and expressed as fold increase (F.I. ± SEM) of each treatment relative to each control. The protein level used was the mean obtained in three separate experiments. CO–: Control; 0.1: 0.1 mg/l SDS; 1: 1 mg/l SDS.

pal non-enzymatic endogenous antioxidant, GSH, was depleted, as demonstrated by the significant increase of its oxidized form, GSSG, especially in hepatopancreas and mantle (p < 0.05) (Fig. 5). Similar to the enzymatic antioxidants, SDS treatment did not cause clear changes in the glutathione balance in gills. 3.4. Hsp70 The treatment with SDS caused an increase of the Hsp70 levels in all analyzed tissues (Fig. 6). In gills the exposure to 1 mg/l SDS resulted in a 1.67 ± 0.09-fold increase, in comparison to the respective control (p < 0.05) (Fig. 6A). In mantle and hepatopancreas, the protein levels increased up to 2.28 ± 0.27 and 1.66 ± 0.24, respectively, with regard to the control (p < 0.05) (Fig. 6B and C).

Fig. 4. CAT activity in M. galloprovincialis tissues (gills, G; hepatopancreas, H; mantle, M) after 18 days of exposure to SDS (0.1–1 mg/l). Data are expressed as unit for ␮g of total proteins (Unit/␮g T.P. ± SEM) and compared to each control. *p < 0.05; **p < 0.01.

4. Discussion The exposure of an organism to xenobiotic substances, leads to alterations of its homoeostasis and an inability to react to the

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environment which surrounds it. The overall toxic effect of SDS is likely due to (a) the foam produced in the aquatic surface, blocking the oxygen exchange primarily by its ability to absorb onto and penetrate the cell membrane; (b) the surfactant head group and a non-polar chain are recognized as toxic to aquatic organisms (Jifa et al., 2005), because they can bind to various macromolecules, including polysaccharides, proteins, peptides and DNA, or insert into various cell fragments (i.e. phospholipids membranes), which may cause inappropriate function. The parameters evaluated in this study cover a broad range of effects, from markers of adaptation to the environment (RVD) to stress and oxidative stress markers. 4.1. RVD The capability of anionic SAAs, as SDS, to penetrate cellular membranes is known to influence the ionic as well as the redox balance of the cells (Grant and Acosta, 1996). Indeed SDS causes a change in the membrane potential by binding to the channel proteins and disrupting the ability of cells to modulate and maintain the ionic balance. Such ability is largely derived from the presence of the anionic polar head which, when the SAA is incorporated into membrane lipids, tends to create an electrostatic gradient which repels other anionic species (Arai et al., 1995), whilst also attracting and increasing the permeability for cationic species (Anderberg and Arturrson, 1993). As reported in Fig. 1 under control conditions (without SDS) the isolated digestive cells of M. galloprovincialis exhibited a partial RVD and similar results were obtained in a previous study (Torre et al., 2013a). In the presence of the detergent the digestive cells of the exposed animals were unable to perform RVD (Fig. 1). The impairment of the homeostatic response was observed in all experimental conditions, even if the digestive cells maintained their viability. In fact, the lysosome stability, evaluated by neutral red retention assay, was not altered in respect to control values (data not shown). SDS showed harmful effects on osmoregulation machinery, likely acting on solute permeability and not on the osmotic water permeability. In fact the initial swelling, due to the entry of water following the osmotic gradient, was about 15% and was identical in both control and SDS treated cells (Fig. 1). It is known that the interaction with lipid membranes can disrupt membrane integrity, thus causing toxic effects (Abel, 1974). The lack of integrity is probably caused by interference with membrane permeability or membrane proteins. One possible cause of membrane integrity disruption is the oxidative stress, leading to a loss of fluidity and increased ion permeability (Livingstone, 2003). In the light of these observations it is conceivable that the impairment of RVD in cells of SDS exposed animals is due to the lack of membrane integrity that does not allow the activation and/or the insertion of membrane transporters involved in ion movement from the cells, leading to osmotic water efflux during RVD. 4.2. ROS production Change in ionic balance causes the alteration of the redox potential of the cell, possibly causing oxidative stress on the organisms (Livingstone, 2003). In our study the treatment with SDS on M. galloprovincialis resulted in an increasing production of ROS in each analyzed tissue (Fig. 2). It has been previously demonstrated that SDS stimulates the production of superoxide ions (O2 = ) through the induction of NADPH oxidase in cell-free systems (Bromberg and Pick, 1985). Recently the effects of SDS as an inducer of ROS production under photo-degrading conditions, have been shown in a study where different SAAs were tested as carriers of photosensitive compounds; compounds dissolved in SDS demonstrated a marked increase of singlet oxygen species production after UV irradiation (Onoue et al., 2008). Some biomarkers of oxidative stress were previously assessed on marine organisms exposed to

SDS, such as fish (Feng et al., 2008; Jifa et al., 2005; Nunes et al., 2008; Sen and Semiz, 2007; Sobrino-Figueroa, 2013), crustaceans (Nunes et al., 2006), mussels (Hoarau et al., 2004; Liu et al., 2010), flatworms (Li, 2008) and plants (Forni et al., 2012). Accordingly, our results demonstrate the close relation between SDS exposure and ROS production, supported by the concentrationdependent increasing trend in the mussel’s hepatopancreas and mantle (Fig. 2). 4.3. Scavenger activity In this study oxidative stress markers, as SOD and CAT activity, as well as GSH depletion have been evaluated, as those represent the major antioxidant defense in mussel tissues (Manduzio et al., 2005) and in all organisms (Benzie, 2000). The SOD activity showed a marked increase in the hepatopancreas and mantle tissues after exposure to SDS (Fig. 3). Perhaps the absence of induction in gills can be explained with an acclimatization process during the chronic exposure, for a tissue which is constantly in contact with the xenobiotic. Similar results have been obtained from Jifa et al. (2005) where chronic exposure of Lateolabrax japonicas to SDS produced an increase in SOD activity in the liver (Jifa et al., 2005). Although the correlation between xenobiotic exposure, oxidative stress and induction of SOD in mussels is well documented (Faria et al., 2010; Vlahogianni et al., 2007; Richardson et al., 2008), in other studies exposure to SDS did not significantly modify the SOD activity (Liu et al., 2010). The induction of SOD is closely related to the production of superoxide radicals which are converted by the enzyme into hydrogen peroxide (H2 O2 ) or peroxide radicals (R–OOH− ) through a dismutation reaction. Thereafter CAT, which is usually located inside peroxisomes, catalyzes the conversion of H2 O2 and peroxides into water and respective alcohol. In our study, results indicate that the induction of CAT was directly dependent upon SOD activity, thus reflecting the production of the substrate for the enzymatic process (Fig. 4). Because SDS caused a marked increase in superoxide species (converted by SOD into peroxides), which represent the ideal substrate for CAT activity, such activity is markedly induced. CAT is a fundamental enzyme during oxidative stress when high levels of ROS are produced; furthermore oxidative stress conditions cause the proliferation of peroxisomes, which are highly efficient in scavenging ROS, especially H2 O2 , which diffuses into peroxisomes from the cytosol (Mittler, 2002). However, acute exposure to a higher concentration of SDS (1 g/l SDS) on Tilapia nilotica caused a marked decrease in CAT activity (Feng et al., 2008) and this difference might be related to exposure modality and the acute action of the compound, under more elevated concentrations. Induction of CAT is, however, usually related to metal exposure (Vlahogianni et al., 2007), organic pollution (Livingstone et al., 1995) as well as environmental factors (Khessiba et al., 2005). Together with SOD and CAT, GSH is one of the most important antioxidant system. GSH is a tripeptide of glutamate (l-Glu), cysteine (l-Cys) and glycine (Gly), and its antioxidant properties depend on the thiol group of the cysteine residue (Manduzio et al., 2005). Under oxidative stress GSH is oxidized to GSSG, and accumulation of GSSG in tissues, thus, represents the evidence of oxidative stress in cells (Circu and Aw, 2010). In this study GSSG concentration displayed an increasing trend (Fig. 5), similar to what was found in SOD and CAT, both in hepatopancreas and mantle (Figs. 3 and 4). Liu et al. (2010) found that exposure to SDS did not substantially modify the GSSG concentration but, in their work, they found a marked induction of glutathione-S-transferase (GST). GST catalyzes the conjugation between GSH and cellular components damaged by ROS and the increasing trend of GST activity was thought by the authors to be a consequence of the continuous input of the anionic surfactant from seawater (Liu et al., 2010).

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Other studies showed an accumulation of GSSG as a consequence of SDS exposure in Gambusia holbrooki (Nunes et al., 2008) and in the yeast Saccaromices cerevisiae (Sirisattha et al., 2004). 4.4. Hsp70 Hsps are a well-studied family of proteins which act as chaperones or co-chaperones in order to reduce the aggregation by holding miss-folded polypeptides and promoting refolding; Hsps are mostly involved in prevention of miss-folding and denaturation of cellular structures, especially under chronic stress, if the normal cellular reducing environment is compromised. (Freeman and Morimoto, 1996). Hsps perform versatile roles in cells, from housekeeping duties under unstressed conditions, including regulation of protein quality control, to life and death decisions following cellular stress via their interaction with members of the apoptotic cell death cascade (Kalmar and Greensmith, 2009). As stated previously one of the biological effects of SDS is the disruption of the protein secondary and tertiary structures caused by the interaction with cationic amino acids residues (Housaindokht et al., 1993) and the consequent induction of oxidative stress (Arai et al., 1995; Grant and Acosta, 1996). In our study the analysis of Hsp70 demonstrated an up-regulation driven by the increase in SDS concentration in the treatment in all the tissues (Fig. 6). For the gills, mantle and hepatopancreas the highest SDS treatment concentration (1 mg/l SDS) resulted in an increase of Hsp70 level. In the literature we have not found evidence of studies involving Hsp70 and SAAs. However, Hps70 is a well demonstrated and efficient marker of stress conditions in mussels as shown by studies on metals (Franzellitti and Fabbri, 2005), pesticides (Joseph and Raji, 2011) and other organic pollutants (Franzellitti et al., 2010) as well as environmental factors (Ioannou et al., 2009). An explanation of our results may lie in the behavior of Hsp70 in the presence of oxidative conditions caused by ROS. In the absence of stress Hsp70 binds monomeric HSF1 (Heat Shock transcription Factor-1) (Morimoto, 1992); under stress, unfolded proteins appear in the cells, Hsp70 releases HSF1 and assists the refolding of damaged proteins. HSF1 can then trimerize and migrate into the nucleus where it activates the transcription of genes encoding Hsps (Kalmar and Greensmith, 2009). Thus, under a chronic condition of stress, an up-regulation of Hsp70 may occur. Furthermore, the depletion of GSH also influences the expression of Hsp70. In a study, Park et al. (2007) found that the depletion of GSH via the inhibition of ␥-glutamyl cysteine synthetase resulted in down-regulation of Hsp70 after 1 day. In our study the results show a depletion of GSH, demonstrated by the increase in concentration of GSSG. However, the differences in the induction of Hsp70 may lie in the exposure modality (Acute vs Chronic). The markers analyzed in this study enabled detection of the influence of a chronic exposure of M. galloprovincialis to SDS. Anionic SAA provoked a chronic oxidative stress, as indicated by the increase in ROS in all the tissues analyzed. The ROS level reached depended mostly on the exposure concentratiob. The activities or levels of scavenger molecules, such as SOD, CAT and GSH demonstrated both the oxidative status and a process of acclimatization, the latter especially in the gills. Finally, Hsp70 was shown to be a sensitive and effective marker of the stress provoked by the ROS in the organism. 5. Conclusions Under the experimental conditions, the results indicate that SDS induces a stress condition that culminates in oxidative stress, demonstrated by the increased production of ROS in all tissues. This situation seems to involve all the endogenous scavenger systems devoted to the elimination of ROS, up-regulating the activity of SOD, CAT, depleting GSH, as demonstrated by the increased levels

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Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis)...
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