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Metallothionein Levels in the Bivalves Callista Chione and Venus

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Metallothionein Levels in the Bivalves Callista Chione and Venus Metallothionein Levels in the Bivalves Callista chione and Venus verrucosa from Two Mediterranean Sites Efthimia Cotou3*, Constantinos Vagiasb, Theodora Raptib and Vassilios Roussisb a National Centre for Marine Research (NCMR), Agios Kosmas, Hellenikon, GR 16604 Athens, Greece. Fax (++301) 9833095. E-mail: [email protected] h School of Pharmacy, Department of Pharmacognosy, University of Athens, Panepistimioupolis Zografou, Athens 15771, Greece * Author for correspondence and reprint requests Z. Naturforsch. 56 c, 848-852 (2001); received February 9/April 9, 2001 Metallothionein, Callista chione, Venus verrucosa Metallothioneins levels (MTs) in the clams Callista chione and Venus verrucosa, collected from two coastal sites in Greece, were determined and quantified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and a spectrophotometric assay (Ellman’s reaction). SDS- PAGE separation in the digestive gland, which represents the hepato-pancreas in clams, de­ monstrated the presence of MTs similar to mammalian MT (rabbit liver Cd, Zn-thionein). No other SH-containing proteins apart from the MTs were detected. MT levels quantified by the Ellman’s reaction indicated seasonal variation for both species. The highest values were recorded in the spring and the lowest in the autumn. The seasonal variation and the differences in the MT levels of the two areas seem to be related to the reproductive cycle of the organisms as well as to abiotic factors of each area. Our results show that both C. chione and V. verrucosa have the potential to be used as biomarkers of metal pollution, provided that the influence of the external factors is safely quantified. Introduction though still controversial has been connected with the homeostasis of essential metals like Cu and In the last decades there has been considerable Zn, detoxification of toxic metals like Cd, Ag and scientific effort to elaborate biological mecha­ Hg and protection against free radicals (Roesi­ nisms or biomarkers in order to assess and moni­ jadi, 1992). tor various contaminants in the marine environ­ The use of MTs as biomarkers of metal pollu­ ment (Kramer Kees, 1994). Among those, tion comes up against the problem of the complex­ metallothionein induction has been suggested as a ity of mechanisms regulating their biosynthesis biomarker of metal pollution in ecotoxicological which is influenced by a wide range of other and biomonitoring studies (George and Olsson, factors like hormones, second messengers, cyto­ 1994). Metallothioneins (MTs) are low-molecular toxic agents and physical stress (Hamer, 1986 weight metal-binding proteins (6-7 kD), first dis­ Kägi, 1991; G erpe et al., 2000). The necessary cri­ covered in the horse kidney cortex (Margoshes teria for species selection as models for metal pol­ and Vallee, 1957). Later, they were described in a lution have truly been met only for a few fish and large number of animal species including mam­ mussels, for other species either too much interfer­ mals, reptiles, amphibians, invertebrates, plants ence and competing sequestration systems are pre­ and microorganisms (Hamer, 1986; Maroni, 1990; sent or MTs studies have not been accomplished Riordan and Vallee, 1991; Carpene, 1993). In yet (George and Olsson, 1994). aquatic invertebrates MTs have been identified in In the present study we investigated the pres­ approximately 50 different species, the majority of ence of MTs in the clams Callista chione and Venm which are mollusks and crustaceans. MTs possess a verrucosa. These species are quite common in high proportion of cysteine residues (30% of total most of the Mediterranean coastal areas. They are amino acids) placed in specific sequences and clas­ found in sand, gravel and mud sea bottoms, dowr sified into three classes according to their homol­ to depths of 30 m (Poppe and Goto, 1993). Both ogy with mammalian. MTs in bivalves generally species sustain commercial fisheries in some areas belong to class I (Roesijadi, 1992). Their role, of the Mediterranean basin such as the southerr 0939-5075/2001/0900-0848 $ 06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen ■ www.znaturforsch.com • E Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. E. Cotouet al. • Metallothioneins inCallista chione and Venus verrucosa 849 Adriatic and the Aegean Sea. Detailed studies on roform were added. The samples were then centri­ their biology are scarce. There is some informationfuged at 6,000 xg for 10 min at 4 °C. Three volumes on the reproductive cycleV. of verrucosa from the of cold absolute ethanol were added to the col­ south Adriatic and Thermaikos gulf (Maranoet lected supernatant. Exceptionally, 1 mg of RNA al, 1982; Galinou-Mitsoudiet al, 1997) and C. chi­ with 40 ^il 37% HC1 was added to the supernatants one from the Gulf of Trieste (Valliet al, 1984), used for the SDS-PAGE electrophoresis (Viar­ but data on metal-binding proteins of these speciesengo et al, 1997). All samples were maintained is lacking. The objectives of this study were toat -20 °C for 1 h, then they were centrifuged at identify and quantify MT levels in these species6.000 xg for 10 min at 4 °C. The supernatants were and compare the MT levels from the two areas.discharged and the metallothionein-containing Two complementary approaches were adopted:pellets were washed with a cold (-20 °C) solution identification of MTs using SDS polyacrylamidecontaining ethanol, chloroform and homogeniza- gel electrophoresis (SDS-PAGE) and quantifica­tion buffer (87:1:12, v/v). A centrifugation at tion using a spectrophotometric assay (Ellman’s6.000 xg for 10 min followed and the pellets were reaction). dried under nitrogen gas. Materials and Methods SDS-PAGE separation Specimens ofC. chione and V verrucosa were The pellets were re-suspended with 50 ^1 of a hand collected by scuba divers and were broughtsolution containingm M 5 Tris, 1 m M EDTA, pH 7. alive to the laboratory. Collection areas were theTo these, 50 fil of 12 mM fluorescent compound thi- Gulf of Elefsis (Neraki) and the Gulf of Chalkisolyte were added. Thiolyte was freshly prepared (Chalia). Care was taken to use individuals of thefrom a 92 m M bromobimane stock solution in ace- same size, since size roughly represents the age oftonitrile. The samples were then incubated in the the organisms. The specimens from each area weredark at room temperature for 30 min. A volume found no more than 100 meters apart from eachof 100 |il 4% SDS was added to the samples. After other. The MT levels were measured in the diges­ incubation in a water bath at 37 °C for 30 min, tive gland of the species based on an adapted pro­200 ^1 of glycerol were added and the samples tocol for estimation of metallothioneins in marinewere stored at -20 °C for one week before the invertebrates (Viarengoet al., 1997). All analytical electrophoresis. The Thiolyte-labelled metallothi­ grade reagents were acquired from Merk, exceptoneins were separated by 10% SDS polyacryl­ those used for the electrophoresis which wereamide gel electrophoresis (Laemmli, 1970) utiliz­ from BioRad. The bromobimane (B-4380), theing 0.025 m Tris pH 8, 0.2 m glycine and 0.1% SDS rabbit liver Cd, Zn-thionein (M-7641) and theas an elecrophoresis buffer. For the electrophore­ RNA (R-7125) were purchased from Sigma. sis we used the PROTEAN II xi Cell apparatus of BIO-RAD set at 80 Volts for the first 15 minutes Sample preparation and at 150 Volts for the following 3:45 hours. After electrophoresis, the gel was maintained in a solu­ All specimens were rapidly dissected. Their di­ tion of methanol:acetic acid:water (45:10:45 v/v). gestive gland was removed and samples of 1 g The fluorescence of the protein bands in the gel (pool of 6-7 glands) were homogenized in a Pot- was evidenced with an UV transilluminator and ter-Teflon homogenizer in three volumes ofm 0.5 photographed on a positive/negative Polaroid 667 sucrose, 20 mM Tris-HCl (pH 8.6) containing film using a Polaroid DS-34 camera. 0.006 m M leupeptine, 0.5m M phenylmethylsulpho- nylfluoride (PMSF) and 0.01% ß-mercapto- ethanol. The homogenates were centrifuged atSpectrophotometric assay (Ellman’s reaction) 30,000 xg for 20 min to obtain a supernatant con­The pellets were re-suspended in 300 [il of 5 mM taining the MTs. The supernatants were treatedTris-HCl buffer, 1 m M EDTA, pH 7. A volume of with ethanol/chloroform solution (Kimuraet al, 4.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid) in 1979). To aliquots of 1 ml supernatant 1.05 ml 0.2of m Na-phosphate buffer, pH 8 was added (Ell- cold (-20 °C) absolute ethanol and 80 (|il of chlo­man, 1958). The MT content was evaluated spec- 850 E. Cotouet al. • Metallothioneins inCallista chione and Venus verrucosa trophotometrically at 412 nm and the metallothi-liver Cd, Zn-thionein (C, E), and from the bovine onein concentration was estimated utilizingserum albumin (H). The MTs extracted from the reduced glutathione (GSH) as the reference stan­clam digestive glands show a relative mobility on dard. The amount of metallothionein was calcu­the gel similar to that of the mammalian standard lated assuming an arbitrary SH content of 21 SH/MT (rabbit liver MT). Known amounts of mam­ mole with a molecular weight of 7 kDa (Mackaymalian Cd, Zn-thionein (5 |il) added in clam sam­ et al., 1993). ples increased the fluorescence of bands C and E.
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