Camp. Biochem. Physiol. Vol. 104C, No. 3, pp. 3555312, 030674492/93 $6.00 + 0.00 0 1993 Pergamon Press Ltd 1993 Printed in Great Britain MINI-REVIEW MECHANISMS HOMEOSTASIS OF HEAVY IN MARINE METAL CATION INVERTEBRATES A. Vr.&nr!Nco*j’ and J. A. NOTT~ *Institute of General Physiology, University of Genoa, Italy; and IPlymouth Marine Laboratory, Citadel Hill, Plymouth, U.K. (Tel. 010-353-8241) zyxwvutsrqponmlkjihgfedcbaZYXW (Received 6 December 1991; accepted for publicarion 3 1 January 1992) Abstract-l. The main mechanisms involved in heavy metal cation homeostasis in marine invertebrate cells are described. 2. Metallothioneins are probably the most important soluble compounds involved in heavy metal cation homeostasis. The biochemical characteristics of these metalloproteins and the relationship between their amino acid composition and heavy metal binding capacity are elucidated. Moreover data are reported concerning the physiological role of metallothioneins e.g. heavy metal detoxification, cellular metal redistribution, free radical scavenger action, etc. The possible meaning of different soluble compounds in heavy metal cation homeostasis is also discussed. 3. The biochemical role of lysosomes in heavy metal cation compartmentalization and the involvement of these organelles in metallothionein accumulation and sequestration are shown. 4. Data are reported concerning the sequestration of heavy metal cations in insoluble granules as a mechanism of metal detoxification. HgII > CuI > Cd11 > CuII > ZnII (Buffle, 1988). The contaminant elements having a very high affinity for protein SH groups can alter the structure and function of these molecules and ultimately affect cell physiology (Webb, 1979). For what concerns essential heavy metals, it is now generally accepted that in cells of living organisms, the concentration of Cu2+ and Zn’+ is not constant but fluctuates within well-defined and restricted ranges. Marine organisms are continuously exposed to variable concentrations of metals in the sea water. This occurs particularly along coasts, which are influenced by anthropogenic heavy metal contamination. In this respect, molluscs, crustaceans and other marine invertebrates are known to accumulate high levels of heavy metals in their tissues and yet survive in these polluted environments (Bryan, 1976). Such tolerance depends on the ability of these animals to regulate the heavy metal cation concentration inside the cell and to accumulate excess metal in non-toxic forms. Three heavy metal cation homeostasis mechanisms have been identified in marine invertebrate cells: INTRODUCTION The term HM refers to transition “d” and post transition elements and therefore the term also comprises the metals in subgroups BI and BII of the periodic table. The present paper will mainly deal with the homeostasis processes of the subgroup BI and BII cations. Among these elements are Zn and Cu, essential oligoelements for cell metabolism as well as Cd, Hg, Ag and Au for which a biological function is not reported. Indeed, all metals produce toxic effects when they penetrate into the cell in excess. Due to their electronic characteristics, the “soft” metals of the subgroups BI and BII have a high chemical affinity for S (S > 0 > N) (Eichorn, 1973). Conversely the “hard” metals which also include essential metals such as K, Na, Ca and Mg have a higher affinity for 0 (0 > N > S). It seems that the biological role of Zn2+ and Cu2+ is particularly related to their high affinity for the sulphydryl groups of enzymes and structural proteins, although they can also react with the N and 0 atoms of nucleophilic groups of nucleotides, nucleic acids, peptides etc. The toxic effects of the non-essential metals are probably caused by their tendency to substitute for CU*+, Zn2+ and compete effectively for binding to biological ligands, although they can also bind them aspecifitally, particularly when the metals are present in the cell at high concentration. Heavy metals are ranked in affinity for SH groups in the following order: 1. Binding to specific, soluble, ligands, the most important of which are metallothioneins; 2. Compartmentalization within membranelimited vesicles mostly recognised as lysosomes; 3. Formation of insoluble precipitates such as Ca/Mg concretions or Ca/S granules. These systems show varying degrees of effectiveness in different organisms and in different cell types of the same organism. TAddress for correspondence: Dr A. Viarengo, Istituto di Fisiologia Generale, Universita di Genova, Corso Europa 26, 16132 Genova, Italy. 355 356 A. VIARENGO and J. A. NOTT ebrates have many peculiar characteristics. In fact, as reported in Table 1, the amino acid composition of Metallothioneins (MT) are a class of inducible marine invertebrate metallothioneins shows differproteins with the following characteristics: they are ences from mammalian ones that could justify the soluble, heat stable, low molecular weight (in mampeculiar biochemical properties of the former metalmals 67000 Daltons; 61 amino acids), sulphydryl loproteins. In general these contain less cysteine than rich and have a particular amino acid composition, mammalian metallothioneins; moreover, the relative marked by a high content of cysteine (20-30%) and amount of various amino acids varies in the metalvirtual lack of aromatic amino acids and histidine or lothioneins from animals belonging to different methionine. phyla. As an example, in mussel metallothioneins the The amino acid sequence has a characteristic disabsence of methionine, a lower serine and higher tribution of cysteinyl residues such as Cys-X-Cys glycine content than in mammalian metallothioneins allowing the formation of typical metal-thiolate cluswas observed. Only few data are, at present, available concerning ters which display specific spectroscopic features. These proteins show a high heavy metal-binding the turnover rate of metallothioneins in marine incapacity (612 gr at/mole thionein) (Kagi and Kovertebrates; however in the oyster Crassostrea jima, 1987). Metallothioneins may bind either essenvirginica Cd-thioneins show a half-life of about 4-5 tial metals Zn and Cu or pollutant metals Cd, Hg, days (Roesijadi et al., 1991) similar to that of mamAg, etc. In this way they detoxify the excess of metals malian metallothioneins (3-6 days) (Bremner, 1987). that has penetrated into the cells (Bremner, 1987). It is now generally accepted that, intracellularly, Indeed, synthesis of metallothioneins can be greatly metallothioneins exist as different isoforms. When the enhanced by the presence of metals, although hormetalloprotein-containing fraction is purified from mones (glucocorticoids) and stress can also stimulate mammalian tissues (Kimura et al., 1979), molluscs the neosynthesis of these metalloproteins (Kagi and (Frazier, 1986; Roesijadi, 1986), crustaceans (Olafson Nordberg, 1979). et al., 1979b) and algae (Olafson et al., 1979a), It was stated at the “Second International Meeting two-three isoforms can be resolved by chromatoon Metallothionein” (Kagi and Kojima, 1987) that graphic separation on a DEAE anion exchange all metalloproteins which show some of the characcolumn. However, results obtained by anion exteristics reported above may be termed metallothchange chromatography (Klauser et al., 1983) or ionein and ascribed to classes I-II-III on the basis of reverse phase HPLC analysis show that metalloththe similarity of their chemical structure to that of ioneins from vertebrate kidney and liver may be mammalian metallothionein (class I). separated into 4-6 isoforms (Klauser et al., 1983; Metallothionein was first described in mammalian Hunziker and Kagi, 1985; Koizumi et al., 1985). kidney cells by Margoshes and Vallee (1957) and Only two metallothionein isoforms have been desubsequently identified in most living organisms tected in oysters (Fowler et al., 1986; Roesijadi et al., (Hamer, 1986). Recent work has demonstrated 1989), although the same author was able to separate that marine invertebrates synthetize metallothionein six isoforms of Hg-MT from mussel tissues by anion having biochemical characteristics and functional exchange HPLC (Roesijadi, 1986). properties similar to those of vertebrates (George et Amino acid sequence analyses of metallothionein al., 1979; Olafson et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA al., 1979b; Frankenne et al., from the mollusc Crassostrea virginica (Roesijadi et 1980; Lerch et al., 1982; Viarengo et al., 1984; al., 1989) show that this molecule is more closely Frazier, 1986; Hamer, 1986; Roesijadi, 1986; Fowler related to vertebrate metallothioneins (Table 2) than et al., 1986; Viarengo, 1989). It is important to point the proteins extracted from a crustacean, namely the out, however, that metallothioneins of marine invertcrab Scylla serrata (Lerch et al., 1982; Otvos et al., METALLOTHIONEINS Table I. Amino Amino l/2 c$5 Asp Thr Ser Glu Pro GlY Ala Val Met Ile Leu TY~ Phe His LVS A.@ acid Mussel* Cd-tbionein 20.0 9.1 6.3 6.5 5.8 3.8 18.6 4.6 4.2 0.0 3.5 3.0 0.0 0.0 0.0 11.5 1.2 Taken from: ‘Frankenne 1IWinge et al., 1981. et al., 1979; acid comvxition Crassostrea u.t Cd-thionein 29.4 7.3 7.3 1.3 4.4 5.9 16.2 5.9 1.5 0.0 1.5 0.0 0.0 0.0 0.0 13.2 0.0 tRoesijadi of marine invertebrate and mammalian Number of residues (%) Sea urchini Homarus a.$ Zn-thionein Cu-thionein 34. I 5.8 6.1 2.9 12.0 I.7 14.0 5.8 2.0 0.0 1.7 2.0 0.0 0.0 0.0 II.1 0.0 et al., 1989; fNemer 25.5 10.7 5.6 13.1 5.3 5.1 14.7 6.6 1.4 N.D 0.7 0.9 0.0 0.0 0.0 8.4 2.1 ef al., 1985; $Brouwer metallothioneins Blue Crab/l Cd-thionein Rat liver11 Cu-thionein 28.5 6.8 4.7 11.7 12.0 8.1 7.4 2.6 2.2 0.5 0.6 1.1 0.0 0.0 1.0 11.2 1.7 30.0 8.5 3.8 15.0 6.3 0.0 7.3 8.1 2.3 1.8 1.7 0.7 0.0 0.3 0.0 14.0 0.0 e! al., 1986; llBrouwer er al., 1984; Heavy metal cation homeostasis Table 2. Amino acid composition of the Cu-thioneins purified from mussel tissues compared to the amino acid composition of the Cu-protein extracted from lysosomes and residual bodies 357 The crustacean two-“three metal cluster” structure is likely to be widespread in invertebrate metallothioneins that, as mentioned above, show a lower Number of residues (%) cysteine concentration (Engel and Brouwer, 1989; Musself Mussel$ Roesijadi et al., 1989). Mussel* Cu-lysosomal Cu-residual body Amino acid Co-thionein protein Studies on the metal dissociation kinetics demonprotein strate that in Zn/Cd-thioneins from Cancer pagurus, l/ZCys 17.4 19.6 19.3 9.6 19.0 17.6 the Zn2+ and part of Cd2+ is rapidly released from the Asp Thr 7.1 6.0 6.1 protein (Engel and Brouwer, 1989). These data indiSer 9.7 12.2 11.3 cate that the two “three metal clusters” in crab Glu 5.5 12.8 13.4 metallothioneins may be considered less stable than Pro 4.7 7.2 8.1 18.2 1.6 1.9 the “three and four metal clusters” of mammalian GlY Ala 8.4 3.1 3.1 metallothioneins. Val 4.0 I.1 0.0 Data for Zn/Cu-thionein indicate that copper is Met 0.0 0.0 0.0 present in the form of Cu(I). When the protein is fully Ile 2.9 2.0 2.3 Lell 1.5 3.8 4.1 saturated with Cu(I), it contains 12 g atom Cu/mol, 0.0 0.0 0.0 Tyr which indicates that the metal cations are arranged Phe 0.0 I.8 2.2 differently in the clusters (Kagi and Kojima, 1987). His 1.3 2.1 1.7 Typically they produce spectrofluorimetric emission 6.2 6.9 7.5 LYS 2.9 2.0 2.6 spectra in the red region (around 565 nm) (Beltramini Arg et al., 1989). Moreover, due to the high affinity of Taken from *Viarengo et al., 1984; tviarengo ef al., 1989; IViarengo et al., 1989. Cu(1) for the SH residues, the complex is stable and the metal is not easily released. It is important to note that the Cu-thionein has distinct chemical character1982) and the echinoderm zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Anthocidaris crassispina istics, including the capacity to produce oxidized (Ohtake et al., 1983; Nemer et al., 1985). In fact all insoluble polymers (Weser and Rupp, 1979; the cysteines of the first 27 residues of the molluscan Beltramini and Lerch, 1982). metallothioneins are aligned with those of the mammalian forms. Possible physiological roles of metallothioneins In mammalian Cd/Zn-thioneins the seven metal atoms (Cd/Zn = 5 : 2) are distributed between two tetraIt has been demonstrated that metallothioneins thiolate clusters (Hamer, 1986; Kagi and Kojima, play a role in cellular metabolism by providing a 1987). Cluster A (four metal cations/eleven cysteine reservoir of cations which are essential for newly residues) is associated with the C terminus (AA 31synthesized apoenzymes. Thus Zn/Cu-thioneins are a 61) and cluster B (three metal cations/nine cysteine source of Zn2+ as they can easily release the metal residues) is associated with the N terminus of the (Hamer, 1986). This does not always seem to be the molecule (AA l-30). Functionally, the two clusters case for Cu2+, which is firmly bound in the form of show different affinities for metal cations. At pH 7 or Cu(1); also, the apoenzymes often require Cu(I1). below, cluster A is the first to be saturated and cluster However, it has been demonstrated that Zn/CuB the first to release the metal: initially Cd’+ is comthioneins extracted from Homarus americanus can plexed in cluster A and subsequently, together with partially reactivate Cu*+ deprived hemocyanin, the Zn’+, it saturates cluster B (Kagi and Kojima, 1987). respiratory pigment that contains Cu(1) (Brouwer The differences in the amino acid composition et al., 1986). between mammalian and marine invertebrate metalFurthermore, it is possible to grow cells which lack lothioneins correspond to differences in their the metallothionein coding gene in a medium supchelation properties. In the crab Scylla serrata plemented with low levels of Cu2+ and Zn2+ (Hamer, Zn/Cd-thionein (Cd/Zn = 16: 1) cluster 1 contains 1986). However, these cells are particularly susceponly three metal cations complexed with 9 SH cys- tible to the toxic effects of excess heavy metals in the teine residues, whereas cluster 2 has the same culture medium. This demonstrates that these prometal/cysteine ratio as cluster B in mammalian teins play a fundamental role in the regulation of free thioneins (Engel and Brouwer, 1989). metal cation concentrations. Table 3. Amino terminal comparison Protein NH,-terminal MTI MT2 Trout primary structure of oyster MTI and MT2 and with trout MT-A and mouse MTI MT-A Mouse MT1 amino acid sequence S DPCNCIETGTCACSDXCPATGCKCGPX SDPCNClETGTCACSDXCPATG------:::.: .::.:.:.. : MDPCECSKTGSCNCGGSCKC&;A&-:_: __:_: :.. :_ MDPNCSCSTGGSCTCTSSCA&i&&-- X indicates presence of unidentified NH,-terminal block and unidentified residue at position 17. The latter is probably a modified amino acid. Colon (:) indicates identical residues; period (.) indicates conservative replacement; comparisons are made with oyster MT. (Modified from Roesijadi er al., 1989). 358 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA A. VIARENGO and J. A. Norr In mussels, stressors such as rapid changes in temperature (Viarengo et al., 1988) can induce an alteration in the level of metallothionein in the cells but they are not as effective in this respect as the increase in the intracellular concentrations of heavy metals (Viarengo et al., 1980). The general trend of the processes by which metallothioneins reduce heavy metal cation cytotoxic effects is now generally accepted. Essential and nonessential heavy metal cations react in part aspecifitally with cellular components to cause toxic effects, and in part are detoxified in vesicles and/or granules. Moreover, cations such as Hg(II), Cu(I), Cd(II), etc. with a high affinity for SH residues, may displace Zn*+ from a metallothionein physiological pool always present in the cells (Viarengo et al., 1985b). In turn, the excess of heavy metal cations, including the Zn*+ released from pre-existing metallothioneins, induces in the nucleus the synthesis of the mRNA coding for metallothioneins (Unger et al., 1991) consequently increasing, at cytosolic level, the neosynthesis of apothioneins. These chelate the heavy metal cations thus reducing their cytotoxic effects (Viarengo et al., 1987). The “buffer effects” of the pre-existing metallothioneins and the role of the “induced metallothioneins” in the reduction of cytotoxic effects of heavy metals was confirmed by numerous experiments. The pre-treatment of mussels with a sublethal concentration of metal able to stimulate metallothionein synthesis renders the animals more resistant to exposure to higher concentrations of heavy metals which are usually lethal. It has been clearly demonstrated that the incoming cations (Hg*+ and Cu*+) displace the less toxic Zn*+ from the pre-existing metallothioneins (Roesijadi and Fellingham, 1987; Viarengo et al., 1988). These results confirm the possible involvement of the pre-existing metallothionein pool in heavy metal cation detoxification. Moreover, it has been demonstrated that in mussels the induction of metallothionein synthesis plays an important role in metal detoxification (Viarengo et al., 1987). Experimental animals were exposed to lSOpg/l of Cd *+ for 9 days and the metal accumulated in the digestive gland at a concentration of 62pg of Cd*+ per gram of tissue, most of it being present in the cytosolic fraction. This loading caused complete destabilization of the lysosomal membranes that, as known (Moore, 1981), produces severe cytotoxic effects. In this situation only 20% of the metal in the cytosol was bound to metallothioneins. However, since Cd*+ has a biological half life of about 5-6 months (Viarengo et al., 1985b) in this mussel tissue, after a detoxification period of 4 weeks in clean seawater, the digestive gland still contained about 60 pg/g. At this time, more than 80% of the Cd*+ in the cytosol was bound to metallothioneins and consequently the lysosomal membranes were as stable as in control animals (Viarengo et al., 1987). It has been proposed recently that metallothioneins may play a role in reducing oxidative stress susceptibility of the cell, acting as oxygen free-radical scavenger (Thornalley and Vasak, 1985). However, at the moment, no confirmation from in uivo experiments is achieved that metallothioneins could play such a role in marine invertebrate cells. Possibly other soluble cytosolic molecules may be involved in heavy metal cation homeostasis. Thus, in molluscs and crustaceans, Zn*+ and Cu*+ could be bound in part to low molecular weight ligands. In green sick oysters from Cu2+ polluted areas, copper was bound to compounds of molecular weight lower than 2000 D; after exhaustive purification, five major copper containing fractions were identified (Fayi and George, 1985) the more important of these being a nitrogen heterocyclic compound not yet fully characterized. Glutathione and glutathione metabolites accounted for 1% and 16% respectively of these ligands; glutathione conjugate may be actively transported across the plasma membranes and, therefore, GSH could represent a potential carrier for the elimination of the metal from the cells. Concerning the possible role of GSH in heavy metal metabolism, it is important to point out that more recent works tend to consider the soluble tripeptide as the first line of defence against heavy metal cytotoxicity. In fact the experimental reduction of GSH concentration strongly enhances the alterations in the cell metabolism induced by heavy metals, thus ultimately leading to the death of the animal (Singhal et al., 1987). In crab hepatopancreas a low molecular weight Zn*+ hgand of about 300 D was extracted. On the basis of analyses by mass spectroscopy and nuclear magnetic resonance it was putatively identified as a dicarboxylic acid with a complex ahphatic backbone. In addition, it was suggested that prostaglandins could, at least in mammalian tissues, play a role in the active transport of Zn*+ (Song, 1987). LYSOSOMES It is well documented that lysosomes are able to accumulate high levels of heavy metals (Sternlieb and Goldfisher, 1976). As known, the lysosomal vacuolar system is mainly involved in the catabolism of both endogenous and exogenous molecules and, in particular, in protein turnover (Glaumann and Ballard, 1987). Heavy metals may alter lysosomal physiology by destabilizing the limiting membranes and affecting the activity of lysosomal hydrolytic enzymes (Mego and Cain, 1975; Viarengo et al., 1981). However, these vesicles accumulate high concentrations of heavy metals in non-toxic forms and thus represent a second detoxification mechanism which operates in marine invertebrate cells (Moore, 1981). Different types of biochemical reactions may be involved in the accumulation of specific metal cations in the lysosomes of the various species in different invertebrate phyla (George et al., 1978; Coombs, 1979; Martoja et al., 1980; George and Viarengo, 1985). However, among the biochemical mechanisms involved in lysosomal metal compartmentalization, the binding of the cations to lipofuscin granules may represent a general pathway for metal detoxification (George, 1983a,b). Lipofuscins are mainly lipid peroxidation endproducts which are accumulated in the lysosomes as insoluble lipoprotein granules. Both endogenous oxidative reactions and the metabolism of organic xenobiotic compounds (and transition metals such as Fe3+ and Cu*+) can lead to the formation of oxygen derived free radicals in the cells (Aust et al., 1985). Heavy metal cation homeostasis These are not always fully detoxified by the intracellular antioxidant systems which include the enzymes superoxide dismutase, catalase and GSH peroxidases, the hydrophilic scavengers, glutathione and ascorbate and the lipophilic scavengers, vitamin E and b-carotene (Diguiseppi and Fridovich, 1984). Undetoxified free radicals may react with membrane polyunsaturated fatty acids and start a complex sequence of lipid peroxidation reactions. This process results in the formation of toxic carbonyl compounds and in the formation of lipid-lipid, lipid-protein, protein-protein cross links which alter the structure and function of cell membranes. These partially oxidized, non-functional membranes are incorporated into the lysosomes, where the undegradable, acidic, lipid peroxidation products accumulate as insoluble lipofuscin granules (Nagy, 1988). In most cells of marine invertebrates lipofuscin granules are usually excreted by exocytosis. In the kidney cells of both control and Cd-exposed mussels, lysosomal lipofuscin granules are involved in the detoxification of heavy metals (George, 1983a). The granules from Cd-exposed mussels contain higher levels of Cd*+ and Zn2+ than those from controls (George, 1983a). In a preparation of lysosomes from unexposed animals 85% of the Zn2+ was immobilized in the granules (George, 1983b). Incubation of the granules with 6SZn and ‘09Cd indicates that they can take up metal cations from solutions by a passive adsorption process. The metal uptake is rapid and the different metal cations bind to similar sites on the granules with different affinities. The binding is relatively weak (stability constant = 5, pKa,,, = 6) and readily reversible and, therefore, the metal is rapidly exchangeable (George, 198313). The granules can only sequester metals which are weakly bound to proteins and other cytosolic compounds. Metals which are strongly complexed in the tetrathiolate clusters of metallothioneins are not affected. During the evolution of secondary lysosomes into residual bodies (tertiary lysosomes), the lipofuscin granules “grow” as they accumulate the peroxidation end products. It seems possible that the metals which are loosely bound to the acidic residues of the surface will become “trapped’ by the additional lipofuscin and sterically prevented from moving in or out of the granule. The metals are therefore detoxified and they are finally eliminated, by the exocytosis of the residual bodies. This process can be interpreted as a general heavy metal homeostasis mechanism, which is potentially present in the cells of all living organisms. It is particularly active, for example, in marine invertebrate kidney cells which often have a lysosomal system which is rich in lipofuscin. It must be said, however, that different cells may have different rates of lipofuscin formation and consequently may show different capacities of lysosomal metal sequestration. Mussel kidney cell lysosomes have a long half-life (George and Viarengo, 1985); the lysosomes in these cells may accumulate high concentrations of lipofuscin and heavy metals, among these also Cd*+ and Zn2+, metals which are not usually able to stimulate the lipid peroxidation process and therefore the lipofuscin formation (Viarengo et al., 1990). Digestive gland cell lysosomes have a rapid 359 turnover; they usually have a limited lipofuscin content and only a minimal amount of Cd*+ is sequestered into the granules to be excreted by exocytosis (Viarengo zyxwvutsrqponmlkjihgfedcbaZYX et al., 1987). It must be pointed out, however, that the amount of Cd*+ eliminated in this way is not negligible; in fact, in mammalian cells (muscle, liver, kidney), where Cd*+ is mostly present in the cytosol bound to thioneins, the biological half-life of the metal is about 10-30 years (Friberg et al., 1986); on the contrary, in the digestive gland cells of mussels, and other marine invertebrates, Cd*+ has a half-life of about 6 months. In this case about l&20% of the metal is present in the particulate fraction (Robinson et al., 1985; Viarengo et al., 1985b; Roesijadi and Klerks, 1989) at least in part probably sequestered in the lysosomal vacuolar system, which is eliminated by exocytosis. Although metallothionein and lysosomes are often considered to be involved in two different processes of heavy metal cation homeostasis, recent work indicates that their functions, at least in the case of copper, are linked. Thus, in mussels exposed to 40 /lg/l cu 2+ for 3 days the metal in the digestive gland is bound initially to different subcellular fractions. About 65% is present in the cytosol where it is partially bound to metallothioneins, the synthesis of which increases in response to the intracellular uptake of metal cations. However, during a 612 day detoxification period, there is a net increase of the copper concentration in the lysosomal fraction, whereas the concentration of the metal bound to thioneins decreases. Finally, the Cu*+ sequestered in the lysosoma1 vacuolar system is eliminated by exocytosis of the residual bodies (Viarengo et al., 1981). In the lysosomes, about 70% of the Cu2+ is bound to insoluble, SH-rich proteins which occur although at much lower concentration also in control animals (Viarengo zyxwvutsrqp RABBIT Cluster zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR A Cluster B I Cluster 2 CRAB Cluster Fig. 1. Postulated structures for the two metal-thiolate clusters in rabbit liver and crab (S. serrata) hepatopancreas metallothioneins as proposed by Otvos et al. (1982). pmmiq metals in diet 0 3 Cd Zn Cu Pb lysosome FeZnCaPS lipofuschin Cd? Fa ‘e l ‘. l metals in faeces Fig. 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Fig. 2. Mechanisms of neavy metal cation homeostasis in different tissues of bivalves. FGF = food, gut, faeces; MBoB = mantle, bodywall, byssus; K = kidney; BM = blood, muscle; G = gill Ballan-Dufrancais et al., 1982, 1985 Bevelander and Nakahara, 1966 Brvan. 1973 Carmichael and Bondy, 1981 Carmichael and Fowler, 1981 Carmichael er al.. 1979 Chassard-Bouchaud, 1983 Chassard-Bouchaud and Hallegot, 1984 Chassard-Bouchaud er al., 1985, 1986, 1989 Coombs and George, 1980 (review) Doyle et al., 1978 Fowler and Gould, 1988 Fowler er al., 1975 Galtsoff and Whipple, 1930 George, 1983a, b George and Coombs, 1977 George er al., 1982 George and Pirie, 1979, 1980 George er a/., 1976, 1977, 1980 George and Viarengo, 1985 George et al., 1978, 1979 Gould ef al., 1985 Hianette. 1980 H&den. 1967. 1969 Lowe and Moore 1979 Martoia and Ma&in. 1985 Martoja er al., 1985 Mauri and Orlando, 1982 Morse, 1987 Nakahara and Bevelander, 1967 Neff, 1972 Overnell, 1981 Pasteels, 1968 Pentreath. 1973 Pirie and ‘George, 1979 Pirie et al., 1984 Rav and McLeese. 1987 (review) Rdesijadi, 1980, 1982, 1986 ’ Roesiiadi et al.. 1982 Roesiadi and Hall, 1981 Ruddell, 197I Ruddell and Rains. 1975 Schulz-Baldes, 1974, 1977 Sunila. 1986. 1988 Takatsuki, 1934 Thomson er al., 1985 Tiffanv. 1982 Tripp; i957 Viarengo er al., 1980, 1981, 1985a,b, 1987, 1988, 1989 Yevich and Yevich, 1985 Yonge, 1926 Scallov FGF metals Clam Au MBoB K Scallops metals Clam Mn, Cd Scallop Cd : Ca, Mg, P, Cr, Cu, Zn, Mn, Cd K Scallop BM, K, FGF, MBoB Mussel U La G Mussel BM. K. FGF metals, Ag/Pb Mussel, oyster metals G Mussel, oyster metals K Clams K Scallop Cu, Cd MBoB Clam Fe, Hg MBoB cu Ovster BM, K Cd, Ca, S, Zn Mussel BM, FGF Fe, Cd Mussel K Ca, Fe, Zn, P, S Mussel G, BM, K, FGF, MBoB Zn, Cd Mussel G, BM, K, FGF, MBoB Mg, Fe, Zn, Mn, Ca, P, etc Mussel, scallop metals K, FGF Mussel G, BM, K, FGF, MBoB Cu. Zn, Cd zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Oyster, mussel K. FGF ScalloD Cu; Cd metals K’ Bivalves FGF, MBoB Fe Mussel Zn, Fe FGF Mussel BM Cd Oyster FGF Scallop Atit Wedge shell Mn concretions :: Bivalves Th MBoB Bivalves Ca MBoB Clam mineralized granules K zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Scallop Th, Fe G Mussel Zn. Mn. Co. Fe MBoB, FGF Mussel granules K Mussel Cu, Zn BM Oyster G, FGF Cd Molluscs Clam, mussel Cu. Hg : Mussel Hg G Mussel Hg MBoB cu Oyster Cu, Zn BM, MBoB Ovster BM. K Pb Mussel Cu, Cd, metals, Ti G, K, FGF Mussel cu BM, FGF Oyster Fe, Cu. Zn G, BM, K, MBoB Oyster concretions K Clam Fe MBoB Oyster Cu. Cd Cu; Cd Fe Musse: Scallop Oyster G, FGF, MBoB FGF, K FGF A. VIARENC~and J. A. NOTT 362 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA c ._ 363 Heavy metal cation homeostasis et al., 1985a). This Cu-protein has been extracted from the lysosomes under reducing conditions in the presence of 1% /?-mercaptoethanol; subsequently it has been purified and biochemically characterized. The main fraction has a low molecular weight (about 6800) and a typical UV absorbance with a maximum at 272 nm. The amino acid composition of the Cuprotein (Table 1) is similar to that of mussel metallothioneins from which it differs mainly for the low glycine content (about 1%) and for the high levels of aspartic and glutamic acid (Viarengo et al., 1989). These results seem to indicate that Zn/Cu-thioneins are internalized into the lysosomes (more or less rapidly depending on the isoform considered) where, at acidic pH, Zn*+ IS released from the protein, the Zn-thiolate complex being unstable at pH 5. In contrast, the Cu-thiolate complex is stable at pH 1. Under oxidative conditions, this Zn deprived Cuthionein tends to polymerize into insoluble molecules by forming disulphide bonds. These proteins have a high content of copper which is complexed in a non-toxic form. This proposal is supported by recent findings that the lysosomes in mussels are sites of oxyradical production (G. Winston, 1991 pers. commun.). These data indicate that the conditions in lysosomal vacuoles are suitable for the polymerization of Cu-thioneins and the “in situ” formation of lipofuscin. Moreover, it has been recently demonstrated that copper exposure results in decreased GSH concentration and enhanced lipid peroxidation (Viarengo et al., 1990). This leads to an accumulation of lysosomal lipofuscin in the mussel digestive gland. It is concluded, therefore, that copper ions induce the synthesis of cytosolic metallothioneins and promote oxidative stress conditions. In the lysosomes, the metal is sequestered in both the Cu-thionein oxidized polymer and in the lipofuscin granules. On the contrary, Cd*+ does not stimulate the lipid peroxidation process and Cd-saturated thioneins do not tend to polymerize into the lysosomes, probably because Zn and Cd-thiolate complexes are both unstable at lysosomal acidic pH. This will cause the hydrolysis of the apoprotein and the metal release to the cytosol to be bound to newly synthesized thioneins. These different biochemical pathways of the Cu-Zn and Cd-Zn metallothioneins could explain why the biological half-life of Cu*+ in the digestive gland of mussels is short at 8-10 days while for Cd it extends to about 5-6 months. zyxwvutsrqponmlkjihgfedcb GRANULES The attempts to explain the role of insoluble concretions in heavy metal detoxification are merely speculative. In fact, the biochemical mechanisms which underlie the incorporation of heavy metal cations into intracellular granules have not been elucidated because the available data in this field is essentially qualitative or semi-quantitative. In many marine invertebrate species there are cells in which metals are accumulated in the form of granules (Mason and Nott, 1981; Brown, 1982; Bouquegneau and Martoja, 1987). Indeed, this mechanism of sequestration in membrane-limited vesicles may represent a general strategy for metal 1 CRUSTACEA ] metals in diet metals metals in faeces zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Fig. 4 Fig, 4. Mechanisms of heavy metal cation hameostasis in different tissues of crustaceans. G = gill; K = kidney; FGF = food, gut, faeces; BM = blood, muscle; BoE = bodywall, exoskeleton Al-Mohanna and Nott, 1986a,b, 1987, 1989 Al-Mohanna et al., 1985 Arumugam et al., 1987 Becker et al., 1974 Bryan, 1967, 1968 Bryan ei al., 1986 Bryan and Ward, 1965 Bubel, 1976 Chassard-Bouchaud, 1985 Corner and Rigler, 1958 Dethlefson 1978 Djangmah, 1970 Djangmah and Grove, 1970 Engel, 1987 Engel and Brouwer, 1987 Hopkin and Nott, 1979, 1980 Icely and Nott, 1980, 19X5 (review) Jennings and Rainbow, 1979 Jennings et al., 1979 Koulish, 1976 Martin J.-L. 1973 Martin J.-L. er al., 1977 Nott er al., 1985 Nott and Mavin, 1986 Overnell and Trewhella, 1979 Papathanassiou, 1985 Papathanassiou and King, 1986 Rainbow, 1985, 1987, 1988 Rainbow and White, 1989 Ray and McLeese, 1987 Thomas and Rizt, 1986 Walker, 1977 Walker ef al., 1975a,b Walker and Foster, 1979 White and Rainbow, 1984, 1986a,b Wong and Rainbow, 1987 Wright and Brewer, 1979 Zatta, 1985 Zuckerlandl, 1960 Cu, Zn, Au, Th, Fe Au, Th CU CaPO, Zn, Cu Zn Mn Cu, Hg, Cd Cu, Zn, Mn, Fe, Cr, Cd, Ag, etc Hg Cd CU CU cu, zn Cu, Zn Pb, CaPO,, Au, Th, S Cu, Fe, Th Cd Cd Zn, P Fe Zn Pb (arylsulphatase) Ca, Mg, P, S Cu, Cd Cd Cd Zn, Cu, Cd, Pb, Fe, Mn, etc. Cu, Zn, Cd Cd Zn Cu, Zn Zn Zn Cu, Zn, Cd Zn Cd Zn, Co, Mn cu Shrimp Shrimp Crab Crab Lobster, crabs, decapods Lobster Lobster Isopod Shrimps Shrimp, barnacle, prawn Shrimp Shrimp Shrimp Crab Crab Crab Amphipod Crab Crab Barnacle Crab Crab Copepod Shrimps Ctab Shrimp Prawn Decapods, crab, barnacles Decapods, amphipod, barnacle Crustaceans Barnacle Barnacle Barnacle Barnacle Shrimp Crab Crab Crab Crab FGF FGF 0, FGF FGF G, K, FGF, BoE BM, G, K, FGF K, FGF, BoE G, BM FGF BoE G, FGF FGF FGF FGF FGF FGF FGF FGF G, FGF, BM FGF, BoE G, FGF BM FGF FGF, BM FGF G FGF G, FGF FGF G FGF FGF FGF FGF G, FGF FGF FGF K, FGF, BM FGF 366 A. VIARENGO and J. A. NO~T cation homeostasis. The granules in the different invertebrate tissues can be classified into three types based on the cytochemical and chemical characteristics: iron rich granules, Cu-S containing granules and Mg/Ca concretions. The ferritin-rich, iron containing vesicles are principally related to Fe metabolism (Brown, 1982). The Cu sulphur granules are an extremely heterogeneous group; it has been shown that in some cases, as in pore cells of molluscs, Cu granules are related to the metabolism of hemocyanin, the respiratory pigment (Martoja et al., 1980; Mason and Nott, 1981) and in the digestive gland of crustaceans they are related to both the blood and the moult cycle (Al-Mohanna and Nott, 1989). However, Cu sulphur containing granules may have an important role as a detoxification mechanism. Thus, in amphipods, shrimps and other crustacea, from Cu-polluted areas, Cu and S are accumulated within particular cells of the hepatopancress as insoluble granules (Icely and Nott, 1980; Al-Mohanna and Nott, 1986a). However, the biochemical processes that underlie the formation of these structures are not known and, therefore, a full functional interpretation cannot be deduced. In polluted situations, a range of metals together with P and S can accumulate in the residual lysosomes of the gastropod digestive cells (Nott and Nicolaidou, 1989a,b). When the mature cells finally disintegrate, the metal-bearing lysosomes remain intact in the lumen of the gut and can be detected in the faeces. These lysosomes can be classified as granules of variable composition and appear to function as a detoxification and excretion system. The magnesium/calcium concretions occur as two forms, namely, the carbonate and the phosphatepyrophosphate (PijPPi) granules. The Mg/Ca carbonate granules are easily dissolved and they contain only the pure MgCaCO, salt (Mason and Nott, 1981; Nott and Nicolaidou, 1989a,b). In molluscs these concretions have potential for buffering the pH of body fluids and affecting the deposition of calcium carbonate which is associated with shell growth and repair (Simkiss and Mason, 1983). The carbonate granules never contain any additional metals. In contrast, the Mg/Ca PijPPi granules can contain Mg, Mn, Zn, Cu, Fe, Co and Ni and appear to be linked with metal detoxification. Moreover, these insoluble granules are often present in cells from which they can be eliminated by exocytosis or complete cell disintegration (Mason and Nott, 1981; Mason et al., 1984; Nott and Nicolaidou, 1989b). Again, little is known about the biochemical mechanisms that underlie the formation of Mg/Ca PijPPi concretions. However, it is possible that Ca*+ATPase activity in the membrane of cytoplasmic vesicles may provide the Ca2+ accumulation and H + extrusion necessary for Ca*+ precipitation in the presence of adequate PijPPi levels (Simkiss and Mason, 1983; Viarengo, 1989). It can be suggested that toxic cations which accumulate in the cells may alter the Ca2+ metabolism primariiy by activating voltage-dependent Cachannels and inhibiting the Ca*+-ATPase activity in the plasma membrane (Viarengo and Nicotera, 1991). An impairment of the main systems involved in calcium homeostasis would lead to an increase in the concentration of free Ca2+ in the cytosol. The excess of Ca*+ is usually compartmentalized in the endoplasmic reticulum vesicles or mitochondria (Carafoli, 1987) but could be also sequestered in membrane limited vesicles as concretions precipitated PijPPi. The toxic metal ions that initially induce the alteration of calcium homeostasis mechanisms could therefore be trapped in the concretions by co-precipitations. Thus, the biochemical aspects of heavy metal detoxification centre on reducing the presence of free cations by sequestering them both in soluble or insoluble forms. The animals involuntarily take up metals from food and water and subsequently transport, store and excrete them in order to maintain a continual state of flux that guarantees concentration of free cations into the cells and fluids. In the cells of the different organs, metal could rise up to extremely high concentration, this depending on the capacity of the cell to accumulate the excess of heavy metal in a “non-toxic form” bound to soluble compounds and/or compartmentalized in membrane bound vesicles or granules. However, it is important to point out that in not all the cells of the organisms the different biochemical processes of heavy metal cation homeostasis show the same degree of activity; therefore in the different organs of the same organism the metal could be differentially accumulated. In order to rationalize the basic systems of metal cation homeostasis with the network of tissues, the sites of detoxification can be superimposed on whole animal diagrams with the aim of clarifying which processes are active in the different organs of various marine invertebrates. This exercise reveals that investigations have been mainly concentrated on bivalve molluscs (Fig. 2), gastropods (Fig. 3) and Crustacea (Fig. 4). As shown, within these animals particular cell types in the various tissues are specialized for metal accumulation and detoxification. As known, the metal concentrations in any compartment are the net results of continual uptake and loss, the rate of which can vary linearly or exponentially, according to the tissue (KefkeS, 1986; Mason, 1988). These processes contribute to whole body metal concentrations which reflect, therefore, a multifactorial flow system of some complexity. Data on the rate of flux, together with information on the role of the molecular mechanisms involved in heavy metal cation homeostasis will probably permit the development of mathematical models to predict the likely toxic effects of metals in different organisms. It is tempting to speculate that the models will serve practical purposes in the management of coastal and estuarine waters. zyxwvutsrqponmlkjihgfedcbaZ REFERENCES Al-Mohanna S. Y., Nott J. A. and Lane D. J. W. (1985) Mitotic E- and secretory F-cells in the hepatopancreas of the shrimp Penueus semisulcatus (Crustacea : Decapoda). J. mar. biol. Ass. U.K. 65, 901-910. Al-Mohanna S. 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