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Mechanisms of cadmium and zinc accumulation in the polychaete Neanthes arenaceodentata
Abstracts
173
Sequim Bay was considered to be relatively unaffected by anthropogenic activities at the time of collection, these cadmium levels could reflect bioaccumulation from low, naturally-occurring environmental concentrations over the course of individual life time. The bioaccumulation and intracellular distribution of cadmium in the kidney and partial characterization of cadmium-binding ligands are described in this study. The results in untreated animals are compared to those obtained from clams exposed to 20 pg liter-' cadmium in the laboratory for 14 days. Analysis of cadmium copper and zinc in whole kidneys of wild clams indicated levels of 207+ 133pg Cd g-X dry weight, 4"6+ 1"1 #gg -1 Cu, and l 1 4 + 5 5 p g g -1 Zn. Analysis of cytosol and associated pellet obtained by homogenization and centrifugation at 80000 x g for 90min, shows that 84% of the total cadmium in the kidney was presented in cytosol. The cytosolic cadmium was associated with a low molecular weight metal-binding protein fraction of about 12-5kDM r, that was also enriched in UV-absorbance at 254nm. These characteristics are consistent with those of metallothionein-like proteins. The cadmium-binding peak was rechromatographed on Sephadex G-75 to remove additional high and low molecular weight non-metalbinding substances, then separated further by high performance liquid chromatography using a TSK DEAE 5 PW anion exchange column. This resulted in the resolution of two major cadmium-binding protein peaks and t w o minor cadmium-binding peaks. These peaks were also enriched in 254 nm absorbance. Additional minor peaks rich in 280 nm absorbance and not associated with cadmium were separated from the cadmium-binding peaks as well. These findings indicate a relatively complex situation, with major and minor naturally-occurring cadmium-binding proteins, which could represent isoforms of the proteins. The cadmium-binding peaks were collected during preparatory-scale isolations, and the major peaks will be further characterized. Examination of the kidneys from cadmium-exposed clams indicated little or no increase in the cadmium concentration and a general overall similarity to the other results described above for 'unexposed' clams with a single exception. In this case, cadmium was also detected on high molecular weight proteins ( > 70 kD) (9.6% of the cytosolic cadmium) and with small molecules ( < 3 k D ) (1-9% of the cytosolic cadmium). The remainder (88"5%) was associated with metallothionein-like proteins.
Mechanisms of Cadmium and Zinc Accumulation in the Polychaete Neanthes arenaceodentata. K E N N E T H D. JENKINS & A N D R E W Z. MASON.
Molecular Ecology Institute, California State University, Long Beach, California 90840, USA.
174
Abstracts
In previous experiments we found linear relationships between the free ion activity of Cd [Cd 2÷] in seawater and the accumulation of Cd by the polycheate Neanthes arenaceodentata. The current series of experiments examines the mechanisms which link [Cd 2+] and Cd accumulation in Neanthes and compares this process with the accumulation of Zn, an essential metal. In initial studies we examined the short-term kinetics of Cd and Zn accumulation to distinguish between simple metal adsorption on the surface of the organism and actual subcellular metal accumulation. The data indicate that the linear accumulation of these metals in Neanthes is primarily a subcellular process. These studies also provided information on the rates of uptake of both Cd and Zn. Experiments were also carried out with metabolic inhibitors to determine if metal accumulation was energy dependent. Iodoacetic acid, an inhibitor of glycolysis, had no effect on Cd or Zn accumulation. However, in the presence of oligomycin, an inhibitor of mitochondrial ATPase activity, the accumulation of Zn and Cd increased. Long-term studies were also carried out to examine the kinetics of Zn and Cd accumulation and determine when organisms reach equilibrium with the environment. Experiments are currently underway to evaluate several models of metal accumulation which are consistent with these data.
Characterization of the Bivalve Ultrafiltration Barrier: What Pollutants Pass Through? M. P A T R I C I A MORSE,* E D G A R M E Y H O F E R , t & W I L L I A M E. ROBINSON.~ *Marine Science Center and Biology
Department, Northeastern University, Nahant, Massachusetts 01908, USA; t Department of Zoology, University of Washington, Seattle, Washington 98105, USA & ~Edgerton Research Laboratory, New England Aquarium, Central Wharf Boston, Massachusetts 02110, USA. Ultrafiltration is the initial step in the formation of primary urine and occurs across a barrier in the pericardial glands of bivalves. The functional flter consists of interdigitating basal protoplasmic extensions of the pericardial gland cells, the podocytes along with the underlying basal lamina. The barrier separates the blood sinus from the urinary spaces. Previous comparative studies of the pericardial fluid and blood of Mercenaria mercenaria indicated that although glucose and lipid levels were similar in both fluids, the protein concentrations were significantly lower in the pericardial fluid than in the blood plasma. Ultrastructural studies of the filtration barrier in Mytilus edulis, Chlamys hastata and Mercenaria mercenaria have further characterized the filter. Opaque electron tracers have shown that proteins of 40 000 Da pass through the barrier. However, large molecules such as ferritin at 400 000 Da are stopped at the basal lamina.
173
Sequim Bay was considered to be relatively unaffected by anthropogenic activities at the time of collection, these cadmium levels could reflect bioaccumulation from low, naturally-occurring environmental concentrations over the course of individual life time. The bioaccumulation and intracellular distribution of cadmium in the kidney and partial characterization of cadmium-binding ligands are described in this study. The results in untreated animals are compared to those obtained from clams exposed to 20 pg liter-' cadmium in the laboratory for 14 days. Analysis of cadmium copper and zinc in whole kidneys of wild clams indicated levels of 207+ 133pg Cd g-X dry weight, 4"6+ 1"1 #gg -1 Cu, and l 1 4 + 5 5 p g g -1 Zn. Analysis of cytosol and associated pellet obtained by homogenization and centrifugation at 80000 x g for 90min, shows that 84% of the total cadmium in the kidney was presented in cytosol. The cytosolic cadmium was associated with a low molecular weight metal-binding protein fraction of about 12-5kDM r, that was also enriched in UV-absorbance at 254nm. These characteristics are consistent with those of metallothionein-like proteins. The cadmium-binding peak was rechromatographed on Sephadex G-75 to remove additional high and low molecular weight non-metalbinding substances, then separated further by high performance liquid chromatography using a TSK DEAE 5 PW anion exchange column. This resulted in the resolution of two major cadmium-binding protein peaks and t w o minor cadmium-binding peaks. These peaks were also enriched in 254 nm absorbance. Additional minor peaks rich in 280 nm absorbance and not associated with cadmium were separated from the cadmium-binding peaks as well. These findings indicate a relatively complex situation, with major and minor naturally-occurring cadmium-binding proteins, which could represent isoforms of the proteins. The cadmium-binding peaks were collected during preparatory-scale isolations, and the major peaks will be further characterized. Examination of the kidneys from cadmium-exposed clams indicated little or no increase in the cadmium concentration and a general overall similarity to the other results described above for 'unexposed' clams with a single exception. In this case, cadmium was also detected on high molecular weight proteins ( > 70 kD) (9.6% of the cytosolic cadmium) and with small molecules ( < 3 k D ) (1-9% of the cytosolic cadmium). The remainder (88"5%) was associated with metallothionein-like proteins.
Mechanisms of Cadmium and Zinc Accumulation in the Polychaete Neanthes arenaceodentata. K E N N E T H D. JENKINS & A N D R E W Z. MASON.
Molecular Ecology Institute, California State University, Long Beach, California 90840, USA.
174
Abstracts
In previous experiments we found linear relationships between the free ion activity of Cd [Cd 2÷] in seawater and the accumulation of Cd by the polycheate Neanthes arenaceodentata. The current series of experiments examines the mechanisms which link [Cd 2+] and Cd accumulation in Neanthes and compares this process with the accumulation of Zn, an essential metal. In initial studies we examined the short-term kinetics of Cd and Zn accumulation to distinguish between simple metal adsorption on the surface of the organism and actual subcellular metal accumulation. The data indicate that the linear accumulation of these metals in Neanthes is primarily a subcellular process. These studies also provided information on the rates of uptake of both Cd and Zn. Experiments were also carried out with metabolic inhibitors to determine if metal accumulation was energy dependent. Iodoacetic acid, an inhibitor of glycolysis, had no effect on Cd or Zn accumulation. However, in the presence of oligomycin, an inhibitor of mitochondrial ATPase activity, the accumulation of Zn and Cd increased. Long-term studies were also carried out to examine the kinetics of Zn and Cd accumulation and determine when organisms reach equilibrium with the environment. Experiments are currently underway to evaluate several models of metal accumulation which are consistent with these data.
Characterization of the Bivalve Ultrafiltration Barrier: What Pollutants Pass Through? M. P A T R I C I A MORSE,* E D G A R M E Y H O F E R , t & W I L L I A M E. ROBINSON.~ *Marine Science Center and Biology
Department, Northeastern University, Nahant, Massachusetts 01908, USA; t Department of Zoology, University of Washington, Seattle, Washington 98105, USA & ~Edgerton Research Laboratory, New England Aquarium, Central Wharf Boston, Massachusetts 02110, USA. Ultrafiltration is the initial step in the formation of primary urine and occurs across a barrier in the pericardial glands of bivalves. The functional flter consists of interdigitating basal protoplasmic extensions of the pericardial gland cells, the podocytes along with the underlying basal lamina. The barrier separates the blood sinus from the urinary spaces. Previous comparative studies of the pericardial fluid and blood of Mercenaria mercenaria indicated that although glucose and lipid levels were similar in both fluids, the protein concentrations were significantly lower in the pericardial fluid than in the blood plasma. Ultrastructural studies of the filtration barrier in Mytilus edulis, Chlamys hastata and Mercenaria mercenaria have further characterized the filter. Opaque electron tracers have shown that proteins of 40 000 Da pass through the barrier. However, large molecules such as ferritin at 400 000 Da are stopped at the basal lamina.