Thomas L. Coombs

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.

MECHANISMS OF IMMOBILIZATION AND DETOXICATION OF METALS IN MARINE ORGANISMS

Current concepts of metal metabolism have been reviewed with a view to understanding the factors that influence the uptake, storage and elimination of essential and pollutant trace metals in aquatic organisms. Examples from our own studies on estuarine molluscs exposed to heavy metals have been used to illustrate these factors. When metal uptake was combined with electron microscope studies, a novel storage and detoxication mechanism was revealed, where formation of vesicles within the cell enclose the metal within a membrane. This prevents contact of excess metal with vital constituents and effectively detoxifies it until eliminated or passed on to other tissues, as required. The generality of this mechanism has been established.

Characterisation of cadmium-binding proteins from mussels, Mytilus edulis (L), exposed to cadmium

Three Cd2+-binding proteins have been purified and partially characterised from the digestive gland of the bivalve mollusc, Mytilus edulis, after exposure to Cd2+. The major protein, which was judged to be pure on polyacrylamide gel electrophoresis, showed many of the characteristics of mammalian metallothionein; having a high -SH content, few aromatic amino acids and a high A250/A280 nm ratio which disappears on acidification. It also contains Zn and Cu, but differs in its higher apparent molecular weight of about 25 000 and high glycine content (12-19%). The two additional Cd2+-binding proteins had lower cysteine contents and different molar proportions of Cd2+, Zn2+ and Cu2+.

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.

THE EFFECT OF SALINITY ON THE UPTAKE OF CADMIUM BY THE COMMON MUSSEL, MYTILUS EDULIS (L.)

ABSTRACT The effect of salinity and osmolarity on cadmium uptake by Mytilus edulis (L.) has been studied. Cadmium accumulation in vivo and influx in vitro is increased by dilution of sea water or decreased osmolarity. The influx is dependent upon the osmolarity and the relationship between cation uptake and cellular volume may be similar to the phenomenon observed in mitochondria.

Effects of high stability iron-complexes on the kinetics of iron accumulation and excretion in Mytilus edvlis (L.)

The effects of small molecular weight complexes with a high affinity for iron on the uptake, accumulation and excretion of iron by the common mussel, Mytilus edulis (L.) have been investigated. Fe(III) complexes of citrate, EDTA and 1,10-phenanthroline increased both the rate of uptake and the total amount accumulated when compared to those for particulate ferric hydroxide in sea water, whereas ferrichrome b and the Fe(III) complexes of aceto- and benzo-hydroxamic acids give a decrease. Increased uptake is, however, compensated for by an increased rate of excretion resulting in an almost constant residence time for this metal. The iron is accumulated principally in the viscera with a smaller but significant proportion in the gills. The effects produced by prior complexation of the iron cannot be correlated with either the strength of binding of the complex to the iron or the exchangeability of the iron with other ligands but may be concerned with the endocytosis, the mechanism of uptake for iron previously shown to occur in Mytilus.

Copper metabolism in the plaice, Pleuronectes platessa (L.)

Copper metabolism in a teleost, the plaice, Pleuronectes platessa (L.) from a natural environment has been studied. Distribution in the various tissues of the metal and of five key copper-dependent enzymes: ceruloplasmin EC 1.16.3.1; Superoxide dismutase EC 1.15.11; tryptophan oxygenase EC 1.13.1.12; cytochrome oxidase EC 1.9.3.1 and monoamine oxidase EC 1.4.3.4 have been determined. The copper distribution was found to be similar to that in mammals with the greatest concentrations in the brain and heart. Distribution of the copper enzymes is also similar to that found in mammals. A preliminary characterization of the copper enzymes showed that plaice cytochrome oxidase has a pH maximum 2 pH units more alkaline than the mammalian enzyme and that plaice tryptophan oxygenase is more sensitive to heat denaturation than the mammalian enzyme. The present data form a base-line against which studies on factors affecting the copper metabolism in a teleost can be assessed.

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.