Journal of Survey in Fisheries Sciences 2(1) 11-20 2015

Antioxidant response of the variegated ( varia) under anthropogenic impact

Sureda A.1*; Tejada S.2; Valencia J.M.3; Box A.4

Received: January 2015 Accepted: March 2015

Abstract The variegated scallop (Chlamys varia L.) is an important economic resource in the aquaculture sector in Spain. The aim of the present study was to determine the oxidative status of C. varia, exposed to anthropogenic activities in waters of the Mallorca Island (Western Mediterranean). Specimens were collected from two locations attending to a different degree of human impact. The first station located in the Arenal, a protected and non-polluted area, and the second site was situated inside the bay of Port d’Andratx, an intensively urbanized tourism-dedicated shore. of similar average size of 3.0 cm were collected and gills were obtained. All enzymatic activities - Superoxide dismutase, glutathione reductase and glutathione-S-transferase – showed significant higher values in Andratx when compared to the non-polluted area. The MDA and nitrite levels were also higher in C. varia from Andratx respect to the Arenal. In conclusion, Chlamys varia from the anthropogenic area is under oxidative stress as it was evidenced by increased antioxidant enzymes, nitrite and MDA levels respect to the non-polluted area.

Keywords: Antioxidant enzyme, Balearic Islands, Biomarker, Oxidative stress, Pollution

1-Research Group on Community Nutrition and Oxidative Stress, IUNICS, University of Balearic Islands, and CIBEROBN (Physiopathology of Obesity and Nutrition), E-07122 Palma de Mallorca, Balearic Islands, Spain. 2-Experimental Laboratory, Research Unit, Son Llàtzer Hospital, IUNICS, Ctra. Manacor km 4, E-07198, Palma de Mallorca, Balearic Islands, Spain. 3-Laboratori d'Investigacions Marines i Aqüicultura (LIMIA), D.G. Medi Rural i Marí, Govern Balear, E-07158 Port d'Andratx, Balearic Islands, Spain. 4-Consell Insular d’Eivissa, Dep. Agricultura, Ramaderia, Pesca, Caça i Cooperació Municipal. Avda Espanya nº49, E-07800 Eivissa, Balearic Islands, Spain. *Corresponding author's email: [email protected] 12 Sureda et al., Antioxidant response of the variegated scallop (Chlamys varia)…

Introduction cellular components through protein The variegated scallop (C. varia L.) is a oxidation, lipid peroxidation and DNA member of a Pectinidae family, which damage (Regoli et al., 2004; Sureda et is highly regarded as a sea product al., 2011). Organisms have developed a among the consumers. C. varia complex network of antioxidant and represents an important economic detoxifying defence mechanisms that resource in the aquaculture sector in scavenge or prevent the generation of Spain. This species is widespread and ROS, detoxify toxins and pollutants and common across the British Isles, the repair or remove the damaged Mediterranean Sea and West Africa molecules (Elias et al., 1999). The (Özvarol and Gökoglu, 2013). The antioxidant system includes enzymes variegated scallop lives on rocky such as superoxide dismutases (SOD), habitats and often in algal holdfasts up catalase (CAT), glutathione peroxidase to about 100 m in depth (Oakley, 2007). (GPx) and glutathione reductase (GR). The pollution of persistent organic In addition, glutathione-S-transferases pollutants and metals along coastal (GST) protect cells against toxicants by areas in the whole world is seriously conjugating the thiol group of the increasing derived from anthropogenic glutathione to a wide range of uses that are harmful to marine xenobiotics resulting in a more organisms. In touristic areas such as in hydrophilic and less active compound Mallorca Island, this touristic (Lyons et al., 2003). development, mainly in coastal areas, Biomarkers are increasingly used has resulted in continuous discharge of tools for the assessment of pollution various organic and inorganic materials effects on marine organisms. In this into aquatic environments (Cajaraville way, antioxidant defences, detoxifying et al., 2000). Bivalve molluscs are mechanisms and oxidative damage classically selected as sentinels in markers are evaluated to estimate the monitoring programs in order to assess pollution in coastal environments. The environmental contamination (Rocher aim of this study was to investigate the et al., 2006; Sureda et al., 2013b). oxidative status of the variegated These shellfish, including scallops, are scallop, C. varia, under anthropogenic able to concentrate contaminants in exposure in waters of the Mallorca their tissues to higher levels than the Island. ones found in the seawater. This capacity is due to their sessile and Materials and Methods filtering habits (Metian et al., 2007; Sample collection and experimental Metian et al. 2009; Carro et al., 2012). procedure Pollutants accumulated in the Chlamys varia specimens were bodies of organisms can increase the collected from two locations along formation of reactive oxygen species Mallorca Island waters during summer (ROS), resulting in oxidative damage to 2010 attending to a different degree of Journal of Survey in Fisheries Sciences 2(1) 2015 13 human impact. The first station located height and width were determined to in the Arenal (western Mediterranean, the nearest 0.01 mm using calipers. The 39º43'55"N 2º43'35"E) is situated in a work has been carried out in accordance protected area where human presence is with the EU Directive 2010/63/EU for controlled and it was considered as a experiments and following the clean non-polluted area. The second site recommendations from the Ethics was situated inside the bay of Port Committee of the University of The d’Andratx (39º03'37"N 2º22'51"E), also Balearic Islands (Spain). in the west coast of Mallorca with an intensively urbanized tourism-dedicated Enzymatic activities beach. Scallops were kept in fine Enzymatic activities were determined in polypropylene mesh bags (2 mm) gills of C. varia with a Shimadzu UV- which were included in a bigger mesh 2100 spectrophotometer at 25°C. (8 mm). Mesh bags were placed in a Superoxide dismutase (SOD) depth ranging 5-8 meters over (pmol/min/mg protein) activity was sand/mud and Posidonia oceanica determined by the degree of inhibition mixed bottoms. However, of the reduction of cytochrome C by independently of the depth of every superoxide anion generated by the site, all bags were always suspended 2- xanthine oxidase / hypoxanthine system 3 meters over the bottom. These bags (Flohe and Otting, 1984). Glutathione had a 10 kg ballast of rock in the reductase (GR) activity (nmol/min/mg bottom and a buoy on the surface. protein) was measured by a Scallops were placed during November modification of the Goldberg and and December and were maintained Spooner (1984) method, in which the into the sea during 18-20 months, until rate of conversion of oxidized reaching an average size of 3.0 cm. glutathione (GSSG) to reduced Scallops from each area (n=8) were glutathione (GSH) was estimated by transported to the laboratory in an monitoring oxidation of NADPH in the aerated container, and before one hour assay system at 340 nm. Glutathione-S- gills were dissected and homogenized transferase (GST) activity was in ten volumes (w/v) of 100 mM Tris– determined at 314 nm using reduced HCl buffer (pH 7.5). Each homogenate glutathione (GSH) and 1-chloro-2,4- was centrifuged at 9000g at 4 C for 15 dinitrobenzene (CDNB) as substrates min and supernatants were recovered (Habig et al. 1974). and immediately used for the biochemical analyses. All assays were Malondialdehyde (MDA) determination performed in duplicate and results were MDA (nmol/mg prot) as a marker of normalized by the total protein content lipid peroxidation was analyzed by a determined with a colorimetric method colorimetric assay kit specific for MDA (Biorad Protein Assay), using bovine determination (Calbiochem, San Diego, serum albumin (BSA) as standard. Shell CA, USA) based on the reaction of 14 Sureda et al., Antioxidant response of the variegated scallop (Chlamys varia)…

MDA with a chromogenic reagent to Student’s t-test. Level of significance yield a stable chromophore with for acceptance was p<0.05. maximal absorbance at 586 nm. Results Nitrite determination All C. varia individuals, collected 18- Nitrite levels (nmol/mg prot) in gill 20 month after mesh bags were placed, homogenates were evaluated by had similar dimensions, concretely detection of the liberated NO by gas- between 3-3.5 cm length and 1-1.2 cm phase chemiluminescence reaction with width in Arenal (non-polluted site), and ozone using a nitric oxide analyzer 2-3.5 cm length and 0.6-1 cm width in (NOA) 280i (Sievers) (Braman and Andratx (with anthropogenic impact). Hendrix 1989). Enzymatic activities in gills of C. varia are shown in Table 1. All Statistical Analysis antioxidant enzymes determined Statistical analyses were performed reported significant higher values in using the SPSS statistical software Andratx when compared to the non- package version 21.0 (SPSS Inc., polluted area. The increase in the Chicago, IL, USA). Significant activities was about 41% for SOD, 51% differences were tested by an unpaired for GR and 58% for GST.

Table 1: Enzymatic activities in gills of Chlamis varia.

Arenal Andratx Pvalue

SOD (pKat/min/mg prot) 12.1 ± 0.3 17.1 ± 1.3 * p<0.001

GR (nKat/min/mg prot) 2.83 ± 0.51 4.28 ± 0.28 * p=0.008

GST ( nKat/min/mg prot) 244 ± 25 387 ± 44 * p= 0.007

Enzimatic activities measured in gills of Chlamis varia from a clean area (Arenal) and a polluted area (Andratx). Significant differences were analysed by an unpaired t-test. p<0.05 was considered statistically significant. Values are expressed as mean ± S.E.M

MDA concentration is shown in Fig. 1. Similar results were also reported when The MDA levels were significant nitrite levels (Fig. 2) were analysed higher in C. varia from Andratx respect (7.65±0.47 nmol/mg prot vs. 5.51±0.16 to the Arenal (17.8±0.7 nmol/mg prot nmol/mg prot, p=0.001). vs. 10.5±0.3 nmol/mg prot, p<0.000). Journal of Survey in Fisheries Sciences 2(1) 2015 15

Figure 1: MDA levels in gills of Chlamis varia from a clean area (Arenal) and a polluted area (Andratx). Significant differences were analysed by an unpaired Students’ t-test. p<0.05 was considered statistically significant. Values are expressed as mean±S.E.M.

Figure 2: Nitrite levels in gills of Chlamis varia from a clean area (Arenal) and a polluted area (Andratx). Significant differences were analysed by an unpaired Students’ t-test. p<0.05 was considered statistically significant. Values are expressed as mean±S.E.M.

Discussion to date, this is the first approach The present results clearly evidenced focused on studying the antioxidant that C. varia collected from an response of C. varia living in an anthropogenic area reported an environment with high anthropogenic increased oxidative stress situation uses. Previous works have analysed the when compared with a clean area. In antioxidant response in members of the fact, it has been described that the same genus, C. islandica and C. farreri, presence of contaminants, due to to several pollutants including releases from anthropogenic activities, polycyclic aromatic hydrocarbons is a source of oxidative stress in (PAHs) and metals (Hannam et al., exposed organisms and could induce 2010; Zhang et al., 2010; Liu et al., variations in antioxidant enzyme 2012; Xiu et al., 2014). The results activities (Livingstone, 2003; Santovito presented in these studies confirmed et al., 2005; Sureda et al., 2011). Until that pollutants are highly accumulated 16 Sureda et al., Antioxidant response of the variegated scallop (Chlamys varia)… in the scallops inducing oxidative stress enzymes using GSH as co-substrate, and an antioxidant response. Some including the antioxidant enzyme authors also have evaluated the glutathione peroxidase, result in the presence and accumulation of metals GSH oxidation to GSSG. GR is the and polychlorinated biphenyls (PCBs) main system maintaining this ratio, in tissues of C. varia evidencing that catalyzing the reduction of GSSG to this species is adequate in order to GSH in a NADPH-dependent reaction biomonitor several types of (Trevisan et al., 2014). This reduction contamination in the field (Bustamante could help to protect from the and Miramand 2005; Gutierrez et al., oxidation. Nevertheless, high enzymatic 2008; Carro et al., 2012). activities did not apparently compensate C. varia living in Andratx were the resulting oxidative damage as it was capable of increasing their ROS evidenced with the higher MDA levels scavenging capacity to protect in scallops from Andratx. themselves from the environmental Consequently, the excessive and stressful situation. However, MDA accumulation of oxidative damage may levels were also increased in these result in a loss of cellular integrity. In organisms evidencing a situation of accordance with the present study, C. oxidative stress. SOD detoxifies ferrari cultured for 30 days in seawater −• superoxide anion (O2 ), the first ROS containing benzo (k) fluoranthene product formed in many biological resulted in an antioxidant response in reactions, mainly within the gills and digestive gland together with mitochondria as by-products of aerobic an increased lipid peroxidation levels metabolism (Kujoth et al., 2005). SOD (Pan et al., 2005). In another study, a activity is generally considered as a significant increase in the superoxide useful biomarker of pollution because anion level and the activity of SOD of its relatively short time response to were evidenced in haemocytes of C. environmental stressors (Nasci et al., ferrari under Vibrio anguillarum 2002). In addition, the glutathione challenge or higher temperature (29°C) redox status is oxidized after an (Wang et al., 2012). exposure to stressful situations (Sureda Nitrite, determined as a marker of et al., 2011; Pena-Llopis et al., 2014). NO production, was increased in gills As consequence of these oxidizing from Andratx compared to the non- conditions, two molecules of GSH are polluted site. Nitric oxide (NO) is an linked by a disulfide bond to form a important signalling molecule which is molecule of GSSG. The maintenance of involved in a broad range of the GSH/GSSG ratio is essential for the physiological processes. Significant normal cell metabolism. The GST increases in NO are mainly synthesised activity requires GSH as substrate, by an inducible isoform of nitric oxide leading to the loss of that GSH. On the synthase. NO is considered to be other hand, the activities of other involved in the immune response, Journal of Survey in Fisheries Sciences 2(1) 2015 17 contributing to pathogen elimination (III) reduction with (Franchini et al., 1995). In this case, chemiluminescence detection. −• produced NO can react with O2 to Analytical Chemistry, 61, 2715- generate a highly reactive intermediate, 2718. peroxynitrite (ONOO-). However, when Bryan, N.S., Fernandez, B.O., Bauer, this molecule is produced in an excess S.M., Garcia-Saura, M.F., level or without immune stimulation Milsom, A.B., Rassaf, T., could contribute to oxidative stress Maloney, R.E., Bharti, A., (Thomas et al., 2008; Sureda et al., Rodriguez, J. and Feelisch, M., 2013a). Moreover, it has been reported 2005. 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