Brian J. S. Pirie

Detoxication of metals by marine bivalves: An ultrastructural study of the compartmentation of copper and zinc in the oyster Ostrea edulis

An investigation of the mechanisms of detoxication of copper and zinc by the oyster Ostrea edulis (L) has been carried out using naturally occurring “green-sick” (contaminated by copper) and unpolluted oysters. Electron microprobe X-ray analysis of tissues in the electron microscope gives direct evidence for the structural compartmentation of copper and zinc in separate, specific, granular amoebocytes. The metals are immobilized in membrane-limited vesicles as different chemical compounds, copper being associated with sulphur and zinc with phosphorus. Chemical analyses of serum and tissues of normal and “green-sick” oysters indicate that (a) Cu and Zn are accumulated independently, (b) the Cu and Zn in the serum, while higher than in the surrounding sea water, are maintained at a 10-fold smaller level than the tissues, (c) toxicity is reduced by active uptake from the serum into granular amoebocytes, where it is further reduced by compartmentation in membrane-limited vesicles. It is calculated that the individual cell types may contain as much as 13,000 ppm Cu and 25,000 ppm Zn.

The kinetics of accumulation and excretion of ferric hydroxide in Mytilus edulis (I.) and its distribution in the tissues

Iron, which occurs in sea water as particulate ferric hydroxide, is accumulated to high concentrations by the common mussel, Mytilus edulis (L.). The kinetics of the accumulation and excretion of iron in Mytilus has been studied using 59Fe-labelled ferric hydroxide and the tissue distribution and identification at the sub-cellular level determined by analytical electron microscopy. Iron-59 accumulates in linear proportion to the sea-water concentration and is found in all tissues; the concentration factors for viscera, kidneys, gills, muscle = mantle is 25: 6: 4: 1, respectively. The particulate iron is taken up by pinocytosis by special epithelial cells in the gills, gut, kidney and possibly labial palps and held in membrane-bound vesicles, unaccompanied by mucus in the case of gills and kidney, but with mucus present in the digestive diverticulum and mid-gut cells. There is no free iron within the cytoplasm. Approximately 30 % of the iron presented to the gut is not absorbed being voided with the faeces. The absorbed iron is exocytosed and then passed on to amoebocytes in the haemolymph for transport to other tissues, a major portion being excreted by transfer to the byssal threads.

Metabolism of zinc in the mussel, Mytilus edulis (L.): a combined ultrastructural and biochemical study

The uptake, transport, storage and excretion of zinc has been studied in Mytilus edulis . Zinc accumulates in the soft tissues in proportion to its concentration in sea water whilst the concentration in the haemolymph is little above that in the environment. Uptake is via the gut, mantle and gills. The zinc is transported from the gills and gut (t½ ≈ 8 days) via the haemolymph, either as a high molecular weight complex or in the granular amoebocytes, to the kidney. Most of the body zinc is present in the granular amoebocytes (which are found in all the body tissues) or in the gut and kidney. The kidney forms the major storage organ for many trace metals, containing 30% of the body zinc and a concentration of about 1000 μg/g. Zinc is localized as insoluble granules in membranelimited vesicles occupying some 20% of the cell volume. Excretion of zinc is by defaecation, exocytosis of the kidney granules into the urine and diapedesis of the amoebocytes. A multicompartmental model for zinc metabolism which correlates the ultrastructural and kinetic data is proposed.

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium: The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.

Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.)

Abstract Excretory granules from the kidney of the scallop, Pecten maximus (L.), have been isolated and purified. Their morphology has been determined by electron microscopy and their elemental composition by electron-probe X-ray micro-analysis. The granules, varying in size from 5–15 gmm diameter, are inorganic in nature and contain Ca, Mn, Zn, and P, equivalent to 75% of their dry weight, with Mg, Cu, Fe, Cd, K, S, and Cl also present in much smaller amounts. The granules show large variations in elemental composition but the sum of the metals is always close to equimolarity with the phosphorus content. Electron microscopy of the kidney tissue showed that there is a gradual development of the granules from lysosomal membranous vesicles to highly mineralized membrane-limited vacuoles or residual bodies, which are eventually excreted as such into the urinary tract.

Characterization of metal-containing granules from the kidney of the common mussel, Mytilus edulis

Abstract The metal-containing membrane-limited granules which make up some 20% of the cellular volume of the kidney cells of the marine mussel, Mytilus , have been purified and partially characterized by chemical and histochemical analysis. They contain an inorganic component (10% of their dry weight), principally iron, zinc and calcium, associated with sulphur phosphorus together with a pigmented organic component which has the histochemical and spectroscopic of lipofuschin. Microscopic and enzymic evidence indicates that the lipofuschin is present as a result of lysosomal degradation and peroxidation of cellular membranes. The granules may thus be regarded as tertiary lysosomes or residual bodies. The ash content of these lysosomes is 10-fold higher than those from mammalian sources. For this reason, Mytilus may be a useful model system for the study of intralysosomal metal accumulation.

Cellular metal distribution in the Pacific oyster, Crassostrea gigas (Thun.) determined by quantitative X-ray microprobe analysis

Abstract Tissues of Pacific oysters, Crassostrea gigas (Thun.), from the metal-rich Derwent Estuary, Tasmania, were examined using electron microscopy and the cellular metal concentrations measured by X-ray microanalysis. These data showed that 1. (1) all tissues contained elevated copper and zinc concentrations when compared with other bivalves (e.g. Mytilus ), 2. (2) with the exception of reproductive tissues, calcium, iron, copper and zinc were present in the tertiary lysosomes, 3. (3) copper and zinc were selectively accumulated in membrane-limited vesicles of blood amoebocytes to cellular concentrations of ≈ 70 mM Cu and 390 mM Zn and 4. (4) these amoebocytes contain > 90% of the body copper and zinc.

Metal-containing blood cells of oysters: ultrastructure, histochemistry and X-ray microanalysis

The ultrastructure, histochemistry and X-ray microanalysis of the metal-containing blood cells in three species of oyster, Ostrea edulis, Ostrea angasi and Crassostrea gigas are described. Granular cells containing both copper (up to 400 mM) and zinc (up to 1 M) are present in all three species. In addition, Ostrea edulis has two other granular amoebocyte types which specifically accumulate either copper (400 mM) or zinc (12 M). In all types of amoebocyte, the metals are stored in membrane-limited vesicles. The ultrastructure and histochemical staining characteristics of the vesicular contents vary according to metal composition. Zinc-containing granules appear basophilic after sulphide treatment and display homogeneous electron density; copper/zinc-containing granules are less dense and show varying degrees of crystallinity; copper-containing granules appear crystalline (however, any acidophilic or basophilic reaction is masked by the dark brown precipitate of copper sulphide).

Effects of cadmium exposure on metal-containing amoebocytes of the oyster Ostrea edulis

Oysters, Ostrea edulis, were exposed to cadmium (0.1 mg l-1) for up to 110 d (in 1982) under laboratory conditions in order to determine the effect of Cd exposure on blood amoebocytes. The results demonstrate that Cd-accumulation does not alter the total Cu and Zn concentrations in gill tissue. There was a decrease in the numbers of metal-containing amoebocytes, and electron microprobe analysis showed that this was largely due to a reduction in numbers of the mixed Cu/Zn-containing cells rather than in Cu-or Zn-containing cells. It is postulated that this response, which may involve the release of metals from amoebocytes into gill tissue, is a generalised stress response of this oyster. No evidence was found for the presence of a specific Cd-containing blood cell or Cd-binding protein in blood cells.

Use of Electron Microscope X-ray Analysis in the Determination of Detoxication Mechanisms for Heavy Metals in Shellfish

ABSTRACT The application of electron microscope x-ray analysis to the investigation of the subcellular localisation of heavy metals in polluted marine shellfish has been discussed. A hitherto unrecognised detoxication mechanism has been demonstrated by this technique.