Cytosolic binding of Cd, Cu, Zn and Ni in four polychaete species

Camp. Biochem. Physiol. Vol. 95C, No. 1, pp. 111-115, 1990 Great Britain

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CYTOSOLIC BINDING OF Cd, Cu, Zn AND Ni IN FOUR POLYCHAETE SPECIES K. D. H. ERIKSEN,* T. ANDERSEN,* J. STENERSEN~and R. A. ANDERSEN$ *Section of Marine Zoology and Marine Chemistry, Department of Biology, University of Oslo, P.O. Box 1064, N-0316 Oslo, Norway (Telephone (02) 45-45-43); TDivision of Molecular Cell Biology, Department of Biology, University of Oslo, P.O. Box 1050, N-0316 Oslo, Norway and IDepartment of Toxicology, National Institute for Public Health, Geitmyrsvn. 75, N-0462 Oslo, Norway (Received 12 October 1989) Abstract-l. The cytosolic binding of Cd, Cu, Zn and Ni in four polychaete species collected from metal-contaminated sediment was investigated by gel-filtration chromatography, affinity studies and atomic absorption spectrometry. 2. There were no major species differences in the binding patterns of Zn and Ni in the four annelids. Zn was mainly associated with high molecular weight proteins (>35 kDa), whereas Ni eluted with components of very low molecular weight (~4 kDa). 3. Cd and Cu were associated with proteins of size IO-20 kDa in the detrivore Chaetozone setosa and the carnivorous Goniudu muculutu. In both species, these proteins had high affinity for ‘Wd added in vitro. 4. In the two sediment-feeding polychaetes, Pectinariabelgicu and Orbiniunorvegicu,Cu, Zn and Ni were associated with components of either high or very low molecular weight. 5. It is suggested that analysis for metallothionein-like proteins may not be satisfactory for monitoring purposes, and that other cytosolic components must be included in such studies.

INTRODUCTION

Many transition metals are essential to marine life and must be obtained by organisms from food or the surrounding water. V, Cr, Mn, Fe, Co, Ni, Cu, Zn and MO all have biochemical, physiological and structural roles in some or all marine invertebrates (Bryan, 1984). A number of enzymes and proteins ubiquitous to all life contain metal-ions, frequently in the capacity of electron-acceptors and -donors. Important in the sequestration of potentially toxic metal ions are metallothioneins (Mts) and other low molecular weight metal-binding proteins (Roesijadi, 1981; Stone and Overnell, 1985). Metallothioneins are cysteine-rich, low molecular weight proteins with high affinity for group IB and IIB metals in the periodic system. Zn- and Cu-thioneins may participate in maintaining homeostasis of the two metals, whereas Cd-thionein has been proposed as one of the most important detoxifying sinks for this metal (Engel, 1987; Webb, 1987). Much attention has been paid to bivalve and crustaceans in studies of such proteins, virtually to the exclusion of other marine invertebrates. The polychaetes are, however, the largest macroinvertebrate group in most marine sediments with respect to both numbers and biomass, and at any depth from littoral to abyssal. The group commonly dominate contaminated and disturbed environments, and many polychaete species are of economic importance as food for benthic fish. In the present work, the binding of Cd, Cu, Zn and Ni in extracts from four polychaete species was investigated. Chaetozone setosa, Goniada maculata, Pectinaria belgica and Orbinia norvegica were collected from sediments in the vicinity of Kristiansand, southem Norway. As a result of industrial discharges the

metal levels in the sediments of the sampling area are elevated, especially Cu and Ni (Noes, 1985). In addition to the above, C. setosa and G. maculata were collected in the Oslofjord, an area moderately contaminated with Cu and Ni, but with higher sediment Cd levels than the former area (Egeberg, 1983). The objectives of the study were to investigate cytosolic binding of metals in different polychaete species both with regard to taxonomic differences and as related to different exposure levels.

MATERIALS AND

METHODS

Collectionof polychuetes Samples were collected in the vicinity of Kristiansand, June 1986 and in the inner Oslofjord, January 1988. Sediment was sampled at each site using a Day grab (Eleftheriou and Holme, 1984). The sediment was washed into a tub and gently agitated with surface-water to suspend particles, the contents were sieved (1 and 5 mm sieves) and the resulting material transferred to white plastic trays, Individuals of the four selected species were removed, rinsed briefly in seawater and frozen in liquid nitrogen (- 196°C). The samples were then stored at -20°C until use. Chemicals The Protein Assay Kit came from Bio-Rad, Richmond, California, and Sephadex G-75 was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The isotope ‘Wd (sp. act. 1332.14 MCi/mg Cd, carrier-free) was bought from New England Nuclear, Boston, Massachusetts, USA. DTNB (5,5’-dithiobis-(2-nitrobenzoic acid)), Trizma base and Trizma-HCl were bought from Sigma Chem. Co., St Louis, USA. Metal standards for AAS (Cd, Cu, Zn, Ni) were obtained from Teknolab A/S, Oslo, Norway. All other chemicals were of analytical grade. 111

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Analyticalmethods Protein in the samples were determined by the method described by Bradford (1976) using a kit from Bio-Rad. The method of Ellman (1959) and Ellman et al. (1961) was used for the determination of sulthydryl-groups. Measurement of the sulfhydryl-content in fractions collected from gelfiltration was performed by adding 100 PL of DTNB solution (39.6 mg DTNB in 10 mL 0.1 M phosphate buffer, pH 8.0) to 1 mL of eluate. The absorbance at 412 nm was then read after mixing and waiting for 2 min. The content of Cd, Cu, Zn and Ni in fractions from gel-filtration were determined using graphite furnace atomic absorption spectrometry with Zeeman background correction (PerkinElmer AAS 5000 Zeeman). Recovered ‘(@Cdin column fractions was determined using an Intertechnique CG 4000 gamma-counter. Treatment of samples of gel-filtration The frozen polychaete samples were weighed and immediately homogenized in ice-cold 5 mM Tris-HCl, pH 8.2 (1:4 w/v). The homogenate was then centrifuged at 20,000 g, 4°C for 30 min, and 250 PL of the resulting supematant (extract) was applied to a small Sephadex G-75 column (1.3 x 26 cm). The applied samples were eluted with the homogenizing buffer at 1 ml/mitt, and the absorbance of the eluate was continuously monitored at 254nm. Two aliquots of each extract were applied to the column, the first incubated with 2 CCLof a “Wd marker solution and the eluted fractions counted for gamma-emission and assayed for sulthydryl-content. Fractions of the second eluate were assayed for the concentrations of Cd, Cu, Zn and Ni. The metal content of the fractions was standardized between samples using the total protein content of the extracts. The preparation of rat liver Mt for use as standards in the experiments is described in detail by Andersen and Daae

(1988). RESULTS AND DISCUSSION Absorbance at 254 run

The elution profiles of material with absorbance at 254 nm characteristically had two peaks, one corresponding to the void volume and one in the low molecular weight region. Only in the case of C. setosa for the Kristiansand site was there a third, intermediate peak. The material in this peak eluted from the column at the same volume as mammalian Mt (Figs la,Sa).

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Cytosolic binding of cadmium

In both Chaetozone setosa and Goniada maculata extracts a Cd-peak was observed for components of apparent M, 15-20 kDa (Figs la,b,2b). In C. setosa this constituted the main peak, whereas three other Cd-containing peaks were observed in G. maculata extracts, two of high molecular weight (>35 kDa) and one of apparent M, 5-7 kDa. Suzuki et al. (1979) and Yamamura et al. (1981) found Cd associated with three main pools in the terrestrial annelid Eiseniafoetida, one of which had properties similar to mammalian Mt. As was observed in G. maculata in the present work, cytosolic Cd in E. foetida was associated with components of both high and low molecular weight. The same pattern was found by Jenkins and Sanders (1986) and Jenkins and Mason (1988) in the polychaete Neanthes arenaceodentata experimentally exposed to the metal and in the marine oligochaete Monopylephorus cuticulatus by Thompson (1982). Morgan et al. (1989) found that all the cytosolic Cd present in the intestine of two oligochaete species was associated with a single pool of Mt-like proteins. As was observed above for the Cd-binding proteins in C. setosa and G. maculata, those characterized by Morgan et al. were of higher apparent M, than normally expected for Mt-like proteins (2427.5 kDa). Nejmeddine et al. (1988) isolated a different low molecular weight, cytosolic, metal-binding protein from the polychaete Nereis diversicolor. This protein eluted from a Sephadex G-75 column close to the same volume as mammalian Mt (apparent M, 10 kDa), but was characterized as definitely nonMt by its amino acid composition (only 0.9% cysteine) and its weak association with metals. In our laboratory we have observed a similar, nonMt protein in the marine gastropod Nassarius reticulatus (Andersen et al., 1989), and further work is presently under way to elucidate whether the low molecular weight Cd-binding proteins observed in C. setosa and G. maculata are Mt-like or similar to the non-Mt proteins in N. diversicolor and N. reticulatus.

Elution

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Fig. 1. Gel permeation chromatography of Chaetozonesetosa extracts, Absorbance at 254nm (-) was monitored continuously. Cd (-a-), Cu (-¤-), Zn (-A-) and Ni (-*-) as determined by graphite furnace AAS. (a) Samples from the vicinity of Kristiansand. (b) Samples from the inner Oslofjord. Purified rat liver metallothionein would elute at 2(r22 mL.

Cytosolic binding of Cd, Cu, Zn and Ni

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Fig. 2. Gel permeation chromatography of Goniadamaculataextracts. See Fig. 1 for text and explanation of symbols. (a) Samples from the vicinity of Kristiansand (Cd was not detectable in the fractions). (b) Samples from the inner Oslofiord. Cytosolic binding of copper

Some Cu was associated with components of high molecular weight in all four species and in Orbinia norvegica this was the main Cu-containing pool (Fig. 4). Most of the Cu present in C. setosa extracts eluted with proteins of apparent M, similar to mammalian Mt (Fig. la). A major amount of the Cu found in G. maculata from the least contaminated site was also associated with this pool (Fig. 2b). In G. maculata and Pectinaria belgica collected from the most contaminated area a substantial part of the Cu present also eluted at a volume corresponding to that of free ions and very low molecular weight components (~4 kDa, cf. Figs 3 and 4). Young (1982) and Young and Roesijadi (1983) observed an inducible, low molecular weight, Cu-binding protein in the polychaete Eudistylia vancouveri. They also found increased Cu content in the high molecular weight pool at elevated exposures to the metal, which is analogous to the situation observed for C. setosa. Studies on the mussel Mytilus edulis have indicated the possible binding of cytosolic Cu by amino acids (Coombs, 1974; Fayi and George, 1981). The former inferred, however, that the major ligand was homarine, whereas the latter concluded that most of the very low molecular weight Cu-binding capacity

probably was contributed by a nitrogen heterocyclic compound. Thus, there seems to be at least three distinct patterns of cytosolic Cu-binding in polychaetes: one where most of the metal present is associated with a low molecular weight protein, possibly Mt-like (as for E. vancouveri, C. setosa and low-exposure G. maculata), the second where significant amounts of cytosolic Cu is associated with components of very low molecular weight (as for P. belgica and high-exposure G. maculata), and, finally, where the bulk of the metal is bound to high molecular weight proteins (as in 0. norvegica). Cytosolic binding of Zn

Most of the cytosolic Zn present in the four species was bound to components of high molecular weight (apparent M, > 35 kDa), presumably reflecting the natural presence of high molecular weight Zn-containing proteins and enzymes (Figs la,2a,3,4). Surprisingly, no Zn was found to be associated with the Mt-sized Cd- and &-binding proteins described above for C. setosa and G. maculata. This is in contrast with Nejmeddine et al. (1988), who found three cytosolic Zn-binding peaks by Sephadex G-75 gel chromatography of N. diversicolor extracts, one of which corresponded to the 10 kDa

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4. Gel permeation chromatography of Orbiniu noruegica extracts. See Fig. 1 for explanation of symbols. Cd was not detectable in the fractions.

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Fig. 5. Gel permeation chromatography of Chaetozonesetosa extracts. Absorbance at 254 nm (-) was monitored continuously. Fractions were counted for gamma-emission (-+-) and analyzed for sulfhydryl-content

(-¤-). (a) Samples from the vicinity of Kristiansand. (b) Samples from the inner Oslofjord. Purified rat liver metallothionein would elute at 2&22 mL.

Cd-binding protein isolated. In addition to the void peak, a second, very low molecular weight, Zn-containing peak was evident in C. setosa, G. maculata and P. belgica. They also obtained a third, very low molecular weight, Zn-containing peak, as did Wong and Rainbow (1984) in the shore crab (Curcinus maenas). The latter described this Zn-binding ligand as a dicarboxylic acid with an aliphatic backbone. Free Zn-ions would also elute at this volume and as Zn has lower affinity than Cd or Cu to Mt (Hamer, 1986), such a Zn-peak may represent metal that has been stripped from Mt or other ligands in the course of preparation and separation procedures. Cytosolic binding of nickel

In all four species investigated, Ni was mainly recovered in the very low molecular weight peak (~4 kDa), but some Ni was also associated with high molecular weight components in C. setosa, P. belgica and 0. norvegica. According to the ‘spill-over’ hypothesis proposed by Winge and Rajagopalan (1972) the toxic effect of a metal will assert itself following a saturation of ‘detoxifying sites’. The presence of Ni in the high molecular weight pool may therefore indicate a toxic manifestation of the metal, as virtually all cytosolic Ni present in the Oslotjord 400

samples (least contaminated) was recovered in the very low molecular weight peak. Bray et al. (1982) also observed that the majority of the cytosolic Ni present in the bivalves Macoma balthica and Rangia cuneata was associated with components of apparent M, less than 6 kDa. Binding of lo9Cd and presence of suljhydryl groups

In P. belgica and 0. norvegica extracts the added ‘09Cdwas mainly recovered in the void volume of the column, as were sulthydryl-groups (not shown). In C. setosa and G. maculata, however, some binding of the tracer Cd was also evident by components of apparent M, 10-20 kDa (Figs 5 and 6). Somewhat elevated levels of thiol-groups were also observed in fractions representing 10-20 kDa, whereas insignificant amounts of thiol-groups were recovered in the very low molecular weight pool in all four polychaetes. The metals present in that peak were therefore presumably either in ionic form or bound to non-sulfhydryl components. CONCLUSIONS

In an earlier study we associated the occurrence of Mt-like proteins with trophic level (Eriksen et al.,

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Fig. 6. Gel permeation chromatography of Goniuda maculuta extracts. See Fig. 5 for text and explanation of symbols. (a) Samples from the vicinity of Kristiansand. (b) Samples from the inner Oslofjord.

Cytosolic binding of Cd, Cu, Zn and Ni

1988) as we were only able to establish the presence of such proteins in the carnivorous species investigated. In the present study, however, low-molecular weight Cd- and Cu-binding proteins were evident in both the carnivorous (G. maculata) and the detritusfeeding (C. setosa) polychaetes, but not in the two sediment-feeding polychaetes investigated. Previous studies have indicated a complexity of metal-binding proteins in marine invertebrates (see reviews by Roesijadi, 1981; Engel and Brouwer, 1984; Suzuki, 1987), and the presented results further indicate major differences between polychaete groups with regard to the identity of such proteins. Levels of Mts or metal-binding proteins in marine organisms have been suggested for use in monitoring metal stress (reviewed by Engel and Roesijadi, 1987). The striking differences observed in Cu binding patterns for G. maculata collected at sites of variable ambient metal levels demonstrates that all ligands should be investigated in such surveys, not only Mt-like proteins. REFERENCES

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