Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae).

Cadmium distribution was studied in different subcellular fractions of gill and hepatopancreas tissues of eastern oysters Crassostrea virginica. Oysters were exposed for up to 21 days to low sublethal Cd concentrations (25 microg L(-1)). Gill and hepatopancreas tissues were sampled and divided into organelle fractions and cytosol by differential centrifugation. Organelle content of different fractions was verified by activities of marker enzymes, citrate synthase and acid phosphatase for mitochondria and lysosomes, respectively. In both tissue types, there was a significant accumulation of cadmium in cytosol reaching 230-350 ng mg(-1) protein. Among organelles, mitochondria were the main target for Cd bioaccumulation in gills (250-300 ng mg(-1) protein), whereas in hepatopancreas tissues, the highest cadmium accumulation occurred in lysosomes (90-94 ng mg(-1) protein). Although 75-83% of total cadmium burden was associated with the cytosol reflecting high volume fraction of this compartment, Cd concentrations in organelle fractions reached levels that could cause dysfunction of mitochondria and lysosomes. Organ- and organelle-specific patterns of cadmium bioaccumulation support our previous in vivo studies, which showed adverse effects of cadmium exposures on mitochondrial oxidation in gills and on the lysosomal system of hepatopancreas. This may have important implications for the development of biomarkers of effect for heavy metals and for understanding the mechanisms of toxic effects of metals.

Linkages between cellular biomarker responses and reproductive success in oysters--Crassostrea virginica.

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May-June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.

Expression of P-glycoprotein in southeastern oysters, Crassostrea virginica.

These studies provide important fundamental information regarding the expression of P-glycoprotein (p-gp) in southeastern oysters (Crassostrea virginica). Using rhodamine transport studies, p-gp activity was detected in newly fertilized embryos. A monoclonal antibody (C219) was used to evaluate p-gp expression in oyster tissues. On the basis of laboratory studies, p-gp expression tended to be higher in gill tissues than mantle tissues, and was generally not related to salinity differences. Seasonal studies were conducted with oysters collected monthly for 1 year from Lighthouse Creek, an unpolluted site. There was a general pattern of higher p-gp expression in the warmer months and lower expression in the colder months. In contrast, total gill protein concentrations decreased during the warmer months and increased during the colder months. These studies indicate that there are seasonal patterns in p-gp expression which may represent an adaptive response to natural stressors associated with summer conditions.

Expression of P-glycoprotein in the gills of oysters, Crassostrea virginica: seasonal and pollutant related effects.

The expression of p-glycoprotein (p-gp) in aquatic organisms has been proposed as a biomarker of pollution exposure. Previous research has provided evidence that p-gp is inducible by organic xenobiotics and that p-gp is overexpressed in mussels from degraded areas. However, seasonal changes in expression at polluted sites has not been described previously. The purposes of these studies were to evaluate the expression of p-gp in polluted and unpolluted sites in oysters (Crassostrea virginica) and to determine if there were seasonal differences. P-gp expression and total protein concentrations were measured seasonally in the gills of southeastern oysters, Crassostrea virginica, at degraded and undegraded sites in Charleston Harbor, South Carolina, USA. At all sites, p-gp expression was generally higher during the warmer months and lower in the colder months. Polluted sites tended to show a decrease in p-gp expression in June and September, suggesting that p-gp inhibition may have occurred. P-gp expression was not significantly related to specific classes of sediment contaminants or to overall sediment contaminant loading. Total gill protein concentrations at all sites were lower during the warmer months and higher during the colder months. In general, all sites tended to show decreased total gill protein concentrations when compared to the control site. Total protein concentrations were significantly related to water temperature, specific classes of sediment contaminants and overall sediment contaminant loads, but there was no relationship with salinity.

The effects of glutathione depletion on reproductive success in oysters, Crassostrea virginica.

Glutathione (GSH) is a ubiquitous tripeptide that functions as a very important modulator of cellular homeostasis, including detoxification of metals and oxyradicals. Therefore, depletion of GSH may predispose organisms to pollutant stress. Reproductively active oysters (Crassostrea virginica) were exposed to buthionine sulfoximine in the laboratory to deplete gonadal GSH. The effects of metal exposures (Cd and Cu) on fertilization and developmental assays were evaluated using gametes from control and GSH-depleted adults. Fertilization success was not affected by GSH status, i.e. the fertilization rates of gametes derived from GSH-depleted adults were the same or slightly higher. However, GSH depletion did increase the susceptibility of developing embryos to metal toxicity, i.e. adverse effects on embryonic development were observed at lower metal concentrations with gametes derived from GSH-depleted adults. These effects may be related to diminished removal of free radicals or increased availability of metals. Whereas sperm penetration of embryonic membranes and fertilization success may be facilitated by free radicals, the persistence of free radicals during subsequent developmental periods may adversely affect differentiation and normal development. GSH probably also plays an important role in scavenging toxic metals and reducing metal interactions with essential developmental processes. These results suggest that parental depletion of GSH may increase the susceptibility of embryos to metal toxicity.

Biomarker studies with juvenile oysters (Crassostrea virginica) deployed in-situ.

Hatchery-reared juvenile oysters (Crassostrea virginica) were deployed in situ for approximately 1 month from mid-May to mid-June of 1996 at sites that were classified as reference, agricultural, suburban, or urban/industrial. Cellular responses (lysosomal destabilization, glutathione concentrations, lipid peroxidation, heat shock proteins, metallothioneins, and multi-xenobiotic resistance proteins) were analysed, and their efficacy as biomarkers of stress was evaluated. Increased lysosomal destabilization, glutathione depletion, increased lipid peroxidation, and induction of heat shock proteins and metallothioneins were observed at many of the polluted sites, but increases in multixenobiotic resistance proteins were not. Significant correlations between sediment contaminants and lysosomal destabilization or glutathione concentrations were observed. Similarly, there were significant correlations between sediment cadmium and copper levels and metallothioneins. Although elevated lipid peroxidation products and heat shock proteins were observed at some of the contaminated sites, there were no significant correlations with contaminants. These studies suggest that lysosomal destabilization and glutathione depletion are sensitive, robust indicators of contaminant stress. Although lipid peroxidation and heat shock protein responses were not correlated with contaminants, they are still regarded as valuable indicators of stress. These studies demonstrate the value of using a suite of cellular biomarkers to identify and characterize stress responses related to anthropogenic perturbations.

Metal-specific induction of metallothionein isoforms in the blue crab Callinectes sapidus in response to single- and mixed-metal exposure.

Metallothioneins (MTs) play an important role in the metabolism of copper and zinc during the molt cycle of the blue crab. In this study we examined the hypothesis that MT expression in crabs is metal specific. Anion-exchange chromatography showed one major ZnCuMT (ZnMT-I) in control crabs, two MT isoforms in cadmium-treated crabs (CdMT-I, CdMT-II), and three forms (CuMT-I, CuMT-II, CuMT-III) in copper-treated animals. Amino acid analysis of the carboxymethylated apo-MTs, purified by reversed-phase HPLC, showed minor differences between ZnMT-I, CdMT-I, CdMT-II, CuMT-I, and CuMT-II, while CuMT-III was unique. After combined exposure to cadmium and copper, four MTs with differing copper/cadmium ratios were observed, equivalent to CdMT-II and the three CuMTs. We conclude that the blue crab has four genes that encode different MTs. Transcription of the CdMT-I gene is induced by cadmium, but inhibited by copper. CuMT-I, CdMT-II, and ZnMT-I may be the products of a single gene responsive to copper, cadmium, and zinc. Expression of the CuMT-II and CuMT-III genes is initiated by copper and not by cadmium. We believe that CdMT-I and CuMT-III are important in detoxification, whereas Zn/CuMT-I and CuMT-II are involved in regulatory functions. These results show the importance of the use of mixed-metal exposures in the study of the molecular mechanisms of metal regulation and function.

Comparative sensitivity of gametes and early developmental stages of a sea urchin species (Echinometra mathaei) and a bivalve species (Isognomon californicum) during metal exposures.

Bioassays were developed using sperm of a sea urchin (Echinometra mathaei), and sperm, embryos, and larvae of a bivalve species (Isognomon californicum). Sea urchin spawning was restricted to only a few months of the year and viability of sperm throughout the year varied from 5 to 75%. Sea urchin fertilization assays were affected by temporal variation in sperm viability. Spawning in this bivalve species occurs year-round and there is little temporal variation in sperm viability. Since stringent sperm:egg ratios are not required for the bivalve embryo and larval assays, these were less affected by variation in gamete quality. The relative sensitivity of the various assays were compared during exposure to three different metal pollutants: cadmium, copper, and tributyltin. Gametes and embryos were relatively resistant to cadmium toxicity, but larvae were very sensitive. With copper and tributyltin, sea urchin and bivalve fertilization assays were the least sensitive; and bivalve growth assays were the most sensitive, followed closely by the bivalve embryo assays. On the basis of sensitivity, ease and time required to conduct the assay, and salinity tolerance, the bivalve embryo assay was recommended as the overall single most reliable toxicity bioassay. However a multispecies, multidimensional approach using sperm fertilization assays as well as embryo assays should be employed, perhaps in a hierarchal manner. Larval growth assays were deemed too tedious and time consuming to be used routinely, but due to their high sensitivity, should still be considered as a valuable comparative tool.