A comparison of metal-binding proteins and cadmium metabolism in the marine molluscs Littorina littorea (gastropoda), Mytilus edulis and Macoma balthica (bivalvia)

Marine Environmental Research 28 (1989) 195-200

A Comparison of Metal-Binding Proteins and Cadmium Metabolism in the Marine Molluscs Littorina littorea (Gastropoda), Mytilus edulis and Macoma bMthica (Bivalvia) W. J. Langston, M. J. Bebianno & Zhou Mingjiang* Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, UK

A BS TRA C T Studies on the processes of Cd accumulation, turnover and storage in marine molluscs have revealed important qualitative and quantitative differences between species. In L i t t o r i n a littorea collectedfrom uncontaminated sites, Cd is predominantO~ bound to metallothionein ( M T)-like proteins. Exposure to Cd. in the laborato O' and.[ield, results in an increase in Cd hound to these proteins, together with limited increases in Cd in the high molecular weight ( H M W) pool (the latter partly a result of haemocTanin-mediated transport ~tcCd in the haemolymph ). Polarographic determination ~['heat-stable M Tlike compounds in L. littorea indicates there is little overall increase in the amount of protein produced in response to Cd exposure, suggesting that much o] the newly incorporated metal ma), be bound to previously svnthesised material. Relatively high levels ~f MT-like proteins, notably in the digestive gland ( the major Cd-storage organ), appear to be an inherent feature ~?/'this .species even in uncontaminated areas. M T-like proteins also sequester much O[the Cd in exposed mussels M ytil us edulis, though, unlike Littorina, protein induction can he quantitatively related to exposure. In contrast, tellinid clams, M a c o m a balthica, Jail to produce MT-like compounds, even in highO' contaminated conditions, and Cd is mainly bound to H M W proteins. These variations in metal-binding behaviour probably explain the large interspec({ic d(ff'erences in experimentally-determined Cd uptake rates and may account for d(fferences in bioaccumulation potential observed in rhefieht. Despite such variabilio', all three .species studied appear to tolerate an it!{tuv ~?/"Cd provided the normal partitioning ~?]metal is not irreversiblt' disturbed. * Present address: Institute of Oceanography, Academia Sinica, 7 Nanhai Road, Qingdao, China.

Marine Environ. Res. 0141 - 1136/90/$03"50 Printed in Great Britain

195 1990 Elsevier Science Publishers Ltd, England.

196

W. J. Langston, M. J. Bebianno, Zhou Mingjiang

Maintaining intracellular homeostasis, albeit at elevated Cd concentrations, probably compensates for the inability of these animals to regulate Cd uptake and.['or their extremely slow rates of Cd excretion.

The induction of sulphydryl-rich MT-like proteins in organisms is often presumed to occur as a result of exposure to environments contaminated with heavy metals. Consequently, such proteins are regarded as potentially specific indicators of metal pollution. It is essential, however, to evaluate their involvement in metabolism, storage and regulation of metals, and preferable to quantify levels of MT, before the value of these indices can be fully exploited. With these objectives in mind we report here results of preliminary comparative studies on the role of MT-like proteins in Cd homeostasis in the gastropod Littorina littorea and bivalves Mytilus edulis and Macoma balthica. Gel-filtration chromatography (Sephadex G-75) was used to separate metal-binding components in cytosolic extracts of molluscs from the field or sampled during exposure experiments (400 #g Cd litre- 1, Littorina, Mytilus; 100#g Cd litre 1, Macoma.1 3 Quantification of thiolic proteins in heattreated cytosol (including column fractions) was accomplished by differential pulse polarography. 4 Cadmium uptake in L. littorea was continuous over 65 days ( 6 . 2 t l g C d g - l d a y 1). Although, overall, this process is essentially irreversible there is some remobilisation of the metal within Littorina. Thus, Cd initially taken up by the gills is transported to other tissues in the haemolymph, possibly bound to haemocyanin. As kidney binding sites reach saturation the metal is increasingly transferred to the digestive gland, the major storage organ for Cd. 2 Accumulation of Cd in the digestive gland is shown in Fig. 1A. The subcellular distribution of Cd in the digestive gland (and other tissuesl of Littorina reveals that the metal is mostly bound to MT-like proteins at unpolluted field sites. Cadmium contamination (laboratory and field) results in a direct increase in Cd associated with both MT and HMW pools (Fig. 1A). However, in contrast to MT, HMW ligands rapidly become saturated with Cd during exposure. Measurement of heat-stable, thiolic proteins in contaminated L. littorea (20"8 m g g 1 in the digestive gland, after 65 days) revealed little overall increase relative to controls (18"5mgg-1) despite the large influx of Cd. These data suggest that most of the Cd is sequestered by relatively high levels of MT-like protein inherent in the digestive gland of this species, and that net MT levels do not increase significantly as a result of de novo synthesis. Protein turnover rates have yet to be determined, however. Cadmium accumulation in M. edulis (12"5 #g g- l day- l), though

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W. J. Langston, M. J. Bebianno, Zhou Ming/iang

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Metal-binding proteins and Cd in marine molluscs

199

marginally faster than in L. littorea, displays similar characteristics with regard to partitioning. Thus, in mussels, MT-Cd increases continuously with time while H M W proteins become saturated at a relatively early stage during exposure (Fig. 1B). Unlike L. littorea, however, MT production in M. edulis is a direct function of Cd exposure (Fig. 2). Metallothionein concentrations increase by a factor of 3, from 2-3 mg g- 1 to approximately 9 mg g- 1, after 30 days' exposure. No significant changes could be detected in controls. A reasonable linear relationship exists between MT and Cd concentrations in M. edulis as described by the equation: MT in mussels (mg g 1) = 0"0045 Cd (gig g- 1) + 3-03 (r = 0"803, P < 0-001) The uptake rate for Cd in clams, Macoma balthica (0"3/~gg-1 day-1) was slow when compared with the above species, even after allowing for the lower exposure concentration. In marked contrast to M. edulis and L. littorea, no evidence could be found of MT involvement in Cd accumulation and storage, either in field- or laboratory-exposed clams. Most of the Cd in M. balthica is bound to H M W proteins, with a much smaller proportion ( < 15%) of cytosolic Cd associated with very low molecular weight ligands. This latter pool is most significant following exposure to Cd and may signify involvement in the elimination of the metal, albeit limited, when intracellular concentrations are high. 3 The ability to synthesise MT-like proteins in response to Cd is clearly not uniform among marine molluscs. The variations in metal-binding behaviour described undoubtedly contribute to interspecific differences in Cd bioaccumulation potential observed in the laboratory and in the field. Thus, mean bioconcentration factors (24h) calculated from experiments with Macoma, Littorina and Mytilus were 3, 16 and 31 respectively while field data (animals collected at a site in Poole Harbour) reveal corresponding differences in tissue burdens of 12, 38"8 and 38'9 gg g- 1 in these species. It is notable that Cd accumulation is an order of magnitude higher in those molluscs which produce MT-like proteins, including Littorina, Mytilus (this study) and oysters, Ostrea edulis, 5 than in non-producers such as Macoma (and related clams, Scrobicularia plana, own unpublished results). Paradoxically then, failure to synthesise MT need not necessarily imply lower tolerance to metals for species like Macoma, since the resulting reduced accumulation rates/permeability characteristic of these clams may ~compensate' for the absence ofa detoxifying protein. Indeed, it is interesting to note that, despite the interspecific variability in metal-binding patterns described here, a moderately high Cd influx is tolerated by all three species studied, provided that normal partitioning of metals is not irreversibly

200

w. J. Langston, M. J. Bebianno, Zhou Mingjiang

disturbed. Acute toxicity is only encountered when this intracellular balance becomes grossly distorted.

ACKNOWLEDGEMENT Support from the Department of the Environment (PECD 7/7/335) is gratefully acknowledged.

REFERENCES 1. 2. 3. 4.

Langston, W. Langston, W. Langston, W. Bebianno, M. 59 64. 5. Frazier, J. M.

J. J. J. J.

8~; Zhou Mingjiang, Mar. Biol., 92 (1986) 505-15. & Zhou Mingjiang, J. Mar. Biol. Ass. UK, 67 (1987) 585 601. & Zhou Mingjiang, Marine Environ. Res., 21 (1987) 225 37. & Langston, W. J., Portugaliae Electrochimica Acta, 7 (1989)

& George, S. G., Mar. Biol., 76 (1983) 55 61.