Involvement of Metallothionein in Zn Accumulation and Elimination Strategies in Ruditapes decussatus A. Serafim, M. J. Bebianno CIMA, Faculty of Marine and Environmental Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Received: 15 November 2005 /Accepted: 1 July 2006 Abstract. The aim of this study was to identify the involve- ment of metallothionein (MT) in Zn accumulation and elimi- nation strategies in different tissues (gills, digestive gland, and remaining tissues) of Ruditapes decussatus. Clams were ex- posed to two Zn concentrations (100 and 1000 lg/L) for 40 days, followed by a depuration period of 50 days. The accu- mulation and elimination processes were complemented by subcellular Zn distribution, to determine the affinity of this element for the different cellular partitions. Subsequently, the involvement of MT was followed. Zinc concentrations in the tissues of R. decussatus exposed to 100 lg/L was partially regulated, whereas in those exposed to 1000 lg/L, Zn was accumulated (exponentially and linearly) throughout the exposure period. The greatest amount of Zn is associated to the insoluble fraction; however, in the highest Zn exposure, 30% is in the thermostable fraction, where MT occurs. Gel filtration chromatography confirmed that the Zn ions accumulated in this fraction were bound to MT. This protein is actively in- volved in the elimination of this metal, through the rapid degradation of the Zn–MT complex because MT and Zn are turning over simultaneously. Therefore, MT participates in Zn homeostasis in R. decussatus when Zn exposure is low. When Zn accumulation increases, the role of this protein changes from a constitutive function to the detoxification of the excess of Zn ions. Zinc (Zn) is an essential oligoelement present in several enzymes and proteins that play an important role in the growth, development, and functioning of all living cells (Prasad 1993). It is involved as a cofactor in numerous metalloenzymes (over 200) and regulatory proteins, including enzymes of both DNA and RNA biosynthesis and repair (Brzóska and Moniuszko-Jakoniuk 2001; VallØe and Auld 1990). Zn also has an important antioxidant role, interacting with the -SH groups in different macromolecules and there- fore preventing their oxidation (Prasad 1993). However, in spite of its essential character, Zn might become toxic above the levels required by organisms (Ballatori 2002). Marine invertebrates developed strategies to survive in the presence of toxic metal levels, by the removal of these com- pounds from the soluble fraction of the cell through different processes, such as cellular excretion (Mason and Nott 1980), retention inside mineralized granules (George and Pirie 1980; Mason and Nott 1980; Nott and Nicolaidou 1993), and binding to metallothionein (MT) and/or other metalloproteins (Langston et al. 1998). The binding of Zn by MT derives from the constitutive function of these proteins able to donate Zn to appropriate molecular receivers (metalloenzymes and transcription fac- tors) and therefore control the activities that depend on this metal, through specific molecular interactions, which are essential for the growth and cellular development (Brzóska and Moniuszko-Jakoniuk 2001; Roesijadi 1994b). The Zn– MT complex is particularly efficient in donating Zn ions to apoproteins of several enzymes that depend on this metal and reestablishing their activity (Udom and Brady 1980). The essential character of Zn implies the existence of homeostatic mechanisms in the organisms, which regulate its absorption, distribution, cellular assimilation, and excretion (VallØe and Falchuk 1993) and this metal seems to be partially regulated by bivalves (Brown and Depledge 1998; Phillips and Rainbow 1993), such as the mussels Mytilus galloprovincialis (Rainbow 1995; Regoli and Orlando 1994), M. edulis (Amiard et al. 1987), Perna viridis (Chan 1988; Chong and Wang 2001; Phillips 1985; Phillips and Rainbow 1993), and Perna perna (Anandraj et al. 2002). Similarly, Zn levels in the tissues of Ruditapes decussatus seemed also to be regulated (RomØo and Gnassia-Barelli 1995; Serafim and Bebianno 2001). Therefore, the use of MT as a biomarker of Zn exposure remains a possibility in some bivalves, such as in R. decussatus. In fact, some field studies revealed that MT reflects the envi- ronmental concentrations of Zn (Hamza-Chaffai et al. 1999, 2000) and others show that MT levels in this clam are inde- pendent of the environmental Zn concentrations (Bebianno and Serafim 2003; Serafim and Bebianno 2001). These results possibly reflect different degrees of environmental exposure levels and regulatory capacity. The aim of this study was to determine the role of MT in Zn accumulation and elimination strategies in different tis- sues of R. decussatus after exposure to two Zn concentrations (100 and 1000 lg/L) for 40 days, followed by a period of Correspondence to: M. J. Bebianno; email: mbebian@ualg.pt Arch. Environ. Contam. Toxicol. 52, 189–199 (2007) DOI: 10.1007/s00244-005-0258-6