Putative antiviral activity in hemolymph from adult Pacific oysters, Crassostrea gigas

Innate, non-specific resistance mechanisms are important to pathogens, particularly for delaying virus replication at the onset of infection. Innate immunity constitutes the first line of defense in vertebrates and is the only one in invertebrates. Little is known about possible antiviral substances in invertebrates. The present work concerns a study of antiviral substances in hemolymph from adult Crassostrea gigas oysters. Despite the detection of cytotoxicity in fresh filtered hemolymph for both mammalian (CC50: 750 microg/ml) and fish cells (CC50: > 2000 microg/ml for EPC cells and 345 microg/ml for RTG-2 cells), an antiviral substance was detected. Fresh filtered hemolymph was capable of inhibiting the replication of herpes simplex virus type 1 in vitro at an EC50 of 425 microg/ml (total proteins) and the replication of infectious pancreatic necrosis virus in EPC and RTG-2 cells at 217 and 156 microg/ml (total proteins), respectively.     

Significance of physicochemical forms of storage in microalgae in predicting copper transfer to filter-feeding oysters (Crassostrea gigas)

Copper distribution has been examined in two microalgae (Haslea ostrearia, Diatom; Tetraselmis suecica, Prasinophyceae) exposed to Cu at 30 microg/L(-1). Exchangeable copper linked at the cell surface was desorbed using 8-hydroxyquinoline-5-sulfonate as complexing agent. Then, incorporated copper was separated between soluble and insoluble fractions. In addition, algae were resuspended in acid solutions, the pHs of which covered the range existing in the digestive tract of bivalves. Considering that the soluble fraction is the most easily transferred in the food chain and that exchangeable Cu is easily desorbed, the percentages of Cu potentially available in microalgae have been assessed. These percentages have been compared with those retained in oysters Crassostrea gigas fed with contaminated microalgae in previous studies. In H. ostrearia, the potentially available fraction of Cu (90%) was very similar to the percentage retained by oysters (93%) when the bivalves were acclimated to this food for 3 weeks. Only half (21%) of the potentially available Cu of T. suecica (42%) was readily assimilated in oysters after 3 weeks. This is in agreement with the results of the desorption tests at physiological pHs which showed that only 15-25% of Cu was lost, despite solubilization of other constituents of T. suecica as demonstrated by the decrease in their dry weight. Bioavailability determined from metal speciation in food allows a relevant prediction of the trophic transfer in the case of H. ostrearia, but caution is recommended in generalizing this mode of assessment as shown in the case of T. suecica.     

Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg)

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.     

Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)

Glutathione is a tripeptide that plays an important role in ameliorating metal toxicity. Depletion of glutathione has been associated with an increased risk of metal toxicity in mammals. An understanding of the toxicological significance of glutathione depletion in oysters would be of considerable importance given the widespread use of bivalves in biological monitoring. Laboratory studies were conducted by using an inhibitor of glutathione synthesis (buthionine sulfoximine) to investigate the effects of glutathione depletion on metallothionein expression, lysosomal membrane destabilization, and lipid peroxidation in Cu-exposed oysters. In oysters exposed to Cu (20 and 80 μg/l) and buthionine sulfoximine (20 mg/l), metallothionein induction was suppressed and cellular stress responses were frequently higher than those observed in oysters exposed singly to Cu. Together, these results suggest that environmental conditions that cause glutathione depletion may increase the potential for adverse effects to pollutants during in situ exposures.     

Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters (Crassostrea rhizophorae)

A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae), using beta-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.     

Acetylcholinesterase and metallothionein in oysters (Crassostrea corteziensis) from a subtropical Mexican Pacific estuary

Substantial efforts have been devoted to developing and applying biomarkers for ecological risk assessment. Bivalve mollusks, such as mussels and oysters, are commonly used in environmental monitoring programs because of their wide geographical distribution, great sensitivity to environmental pollutants, and ability to accumulate anthropogenically derived chemicals at a high rate. Acetylcholinesterase (AChE) activity and metallothionein (MT’s) content are representative specific biomarkers that indicate the presence of anticholinesterasic compounds (like organophosphorus and carbamate pesticides) and metals, respectively. The aim of this study was to evaluate AChE activity and MT’s content in Crassostrea corteziensis from Boca de Camichín estuary. The results obtained here showed that AChE activity was 65% lower in oysters from Boca de Camichín than in control organisms. In contrast, MT’s content in collected organisms was not statistically different from that in control organisms. AChE activity and MT’s content in oysters could be used as early biomarkers of effects and exposure to pesticides and heavy metals, respectively, in aquatic environments.     

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.     

The effects of elutriates from PAH and heavy metal polluted sediments on Crassostrea gigas (Thunberg) embryogenesis,larval growth and bio-accumulation by the larvae of pollutants from sedimentary origin

The release, bio-availability and toxicity of contaminants, when sediments are resuspended have been examined, studying concurrently their effects on the embryogenesis and on the larval growth of the Crassostrea gigas larvae and their bio-accumulation in those organisms. Three characteristic sediments have been selected (one contaminated by PAHs, a second by heavy metals and the last by the both pollutants). The organisms were directly exposed to elutriates obtained from each sediment or fed on algae (Isochrysis galbana) contaminated with the same elutriates. The elutriates used in this study show contamination levels similar to those observed in some polluted coastal and estuary environments. The larval growth test has appeared to be more sensitive that the embryotoxicity test. The biological effects and the contaminant bio-accumulation were more pronounced when larvae were directly exposed to different elutriates. In the case of PAHs, the contamination of algae was sufficient to lead to effect on the larval growth of the Crassostrea gigas. In all cases, a fraction of contaminants adsorbed on suspended particles was bio-available and accumulated by the larvae. This study has shown that resuspending polluted sediments constitutes a threat to pelagic organisms and than the C. gigas larval growth may be proposed as a test to protect the most sensitive areas.     

Relationships between tissue contaminants and defense-related characteristics of oysters (Crassostrea virginica) from five Florida bays

Evidence linking bivalve defense responses with pollutant exposure is increasing. Contaminant effects on immune or defense responses could influence the ability of an organism to resist infectious disease. This study explored relationships between xenobiotic chemicals accumulated in oyster (Crassostrea virginica) tissue and various measures of putative oyster internal defense activities and physiological condition. Defense-related and physiological measurements were made on individual oysters collected from 22 sites at five Florida bays and pooled oyster tissue from each site was analyzed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and certain pesticides. Chemical concentrations, physiological condition, and hemocyte and hemolymph characteristics varied across bays and among sites within a bay. Within-bay comparisons showed that sites with high oyster defense-related activities often had accompanying high tissue concentrations of one or more classes of xenobiotic chemicals. Correlation analysis performed across bays demonstrated significant positive relationships between most defense-related characteristics and at least one contaminant, including various PAH, PCB and trace metal analytes. In combination with other recent studies, these results strengthen the hypothesis that certain xenobiotic chemicals may be associated with elevated oyster hemocyte activities, even though the ultimate influence on disease resistance remains unknown.     

Greater hemocyte bactericidal activity in oysters (Crassostrea virginica) from a relatively contaminated site in Pensacola Bay,Florida

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumulation in both experimental and long-term field exposures. In an effort to elucidate these relationships, 40 oysters were collected from Bayou Chico (BC) and East Bay (EB) in Pensacola Bay, FL, two estuaries known to differ in the type and magnitude of chemical contaminants present. Oyster tissue concentrations of metals, tri- and dibutyltin (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HCs), hemocyte bacterial killing indices (KI), serum lysozyme (LYS) and serum protein (PRO) levels. Average HC, KI, LYS and PRO were significantly higher in BC oysters, which also had significantly higher tissue concentrations of total trace metals, butyltins (BTs), PAHs, PCBs, pesticides, and Mn, Cu, Zn and Sn. EB oysters had low organic contaminant levels and no detectable BTs, but significantly higher concentrations of Al, Cr, Fe, Ag, Cd, and Hg. Simple correlation analysis between specific defense measurements and specific chemical analytes showed specific positive relationships that corroborated previous findings in other FL estuaries. Canonical correlation analysis was used to examine relationships between defense measurements and tissue metals using linearly combined sets of variables. Results were also consistent with previous findings-the highest possible canonical correlation was positive: r=0.864, P<0.0019 among canonical variables composed of HC, KI and LYS for defense, and Fe, Cu, Ag, Cd, Sb, Sn, Ni, Pb and Hg for metals.     

Cloning of a metallothionein gene and characterization of two other cDNA sequences in the Pacific oyster Crassostrea gigas (CgMT1)

Metallothionein (MT) genes encode essential metal-binding proteins involved in metallic homeostasis and detoxification in living organisms. Here, we describe the structure of the first Pacific oyster Crassostrea gigas metallothionein (CgMT1) gene and the sequences of two other MT cDNA. The CgMT1 gene sequence contains three coding exons plus a 5' entirely non-coding exon, and the predicted protein contains 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The three cDNA sequences present few substitutions in either coding sequence or UTRs. Induction of these MT-mRNA in heavy metal-treated oysters (i.e. cadmium) was confirmed by Northern blot analysis and RT-PCR and suggests a potential specific tissue expression rate. Southern blot analysis suggested the presence of multiple CgMT genes, and allowed the detection of restriction fragment length polymorphisms (RFLPs). Although the CgMT1 coding sequence showed 30-73% nucleotide identities with known sequences in other mollusks, it included the specific motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X-X-X-Cys-X-Cys-X(5)-Cys-X-Cys-Lys found in Mollusk family 2. Marine bivalves are commonly used as pollution bioindicators, thus the development of genetic markers based on CgMT1 polymorphism will allow a monitoring of heavy metal exposure in anthropogenically disturbed ecosystems.     

Geostatistical analysis of DNA damage in oysters, Crassostrea virginica, in Lavaca Bay, Texas

This study evaluated the health of the marine ecosystem in Lavaca Bay, Texas using the Eastern oyster (Crassostrea virginica) as the sentinel species. Lavaca Bay has a history of having gradients of concentrations of pollutants present with some areas containing concentrations high enough to pose a threat to marine ecosystem health. The Comet assay was used to evaluate for the presence of genotoxic response in oyster hematocytes. Bayesian geostatistical analysis was then used to determine if the DNA damage in oyster hematocytes was spatially oriented and to develop continuous surface maps of the risk of DNA damage in this sentinel species. Results indicated that proximity to industrial facilities increased the locational risk of genotoxicity in this species.

Effects of cadmium on aneuploidy and hemocyte parameters in the Pacific oyster, Crassostrea gigas

Pacific oysters, Crassostrea gigas, are commonly reared in estuaries where they are exposed to anthropogenic pollution. Much research has been made on the toxicity of cadmium to aquatic organisms because the compound recurrently contaminates their environment. Our study examined the influence of cadmium on aneuploidy level (lowered chromosome number in a percentage of somatic cells) and hemocyte parameters in C. gigas at different stages of life. Adults and juveniles were exposed to two different concentrations of cadmium. The first concentration applied was equivalent to a peak value found in Marennes-Oléron bay (Charente-Maritime, France; 50 ngL(-1)) and the second was 10 times higher (500 ngL(-1)). Exposure to 50 ngL(-1) cadmium caused a significant decrease in the survival time of C. gigas, but exposure to 500 ngL(-1) surprisingly affected the survival time positively. Significant differences in aneuploidy level were observed between the cadmium treatments and the control in adults but not in juveniles or the offspring of the adult groups. The effects of cadmium on hemocyte parameters were analyzed by flow cytometry. Several hemocyte parameters increased significantly after 21 days of cadmium exposure and subsequently decreased. Phenoloxidase-like activity, evaluated by spectrophotometry, varied over the time of the experiment and increased after 66 days of contact with 500 ngL(-1) cadmium. Taken together, cadmium at environmentally relevant concentrations seems to have only moderate effects on aneuploidy and hemocyte parameters.     

对戒毒模式的思考

我国现行的戒毒模式主要包括强制戒毒、劳教戒毒和自愿戒毒.目前全国共有强制戒毒所554个,床位近12万张.13年来累计收治185万人次;劳教戒毒所129个,床位14万多张,每年累计劳教戒毒13万人次;自愿戒毒医疗机构248个,床位近1万张,每年收治近1万人[1].作者从事戒毒临床工作多年,看到戒毒学员反复进进出出戒毒所,痛心之余,深切感受到戒毒事业任重而道远.本文主要针对当前的脱毒状况、复吸问题、戒毒模式进行思考,提出了一种具有中国特色的,可操作性强的综合戒毒模式.     

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 in vitro biotransformation of 2-aminofluorene in the visceral mass of the Pacific oyster,Crassostrea gigas

1. The in vitro biotransformation of 2-aminofluorene (2-AF) was examined in visceral mass microsomes from the Pacific oyster Crassostrea gigas.

2. The major metabolite was N-formyl-2-aminofluorene with smaller quantities of N-hydroxy-2-aminofluorene and nitroso-2-aminofluorene or 2-nitrofluorene.

3. The mechanism of N-oxidation was explored using various inhibitors, stimulators, cofactors, and pH modifications within incubations.

4. N-Oxidation may occur via a one electron mechanism which involves a transition metal and/or lipid peroxidation.

5. Flavin-containing monooxygenase and possibly prostaglandin synthase also seem to play a role in N-oxidation.     

The in vitro biotransformation of 2-aminofluorene in the visceral mass of the Pacific oyster, Crassostrea gigas

1. The in vitro biotransformation of 2-aminofluorene (2-AF) was examined in visceral mass microsomes from the Pacific oyster Crassostrea gigas. 2. The major metabolite was N-formyl-2-aminofluorene with smaller quantities of N-hydroxy-2-aminofluorene and nitroso-2-aminofluorene or 2-nitrofluorene. 3. The mechanism of N-oxidation was explored using various inhibitors, stimulators, cofactors, and pH modifications within incubations. 4. N-Oxidation may occur via a one electron mechanism which involves a transition metal and/or lipid peroxidation. 5. Flavin-containing monooxygenase and possibly prostaglandin synthase also seem to play a role in N-oxidation.     

Multi-endpoint toxicities on Chinese rare minnow (Gobiocypris rarus) fed with different diets

To compare the effects of three diets, rare minnow was fed with three diets from 30 dph to mature period. The activities of EROD, PROD, SOD and GST were measured in the WBHs as well as Vtg and TBARS concentrations at 60 dph. The rest fish were fed until adulthood for breeding studies. The group A served as the control group. It was found that Vtg, GST and EROD were significantly increased in the groups B and C, but SOD, TBARS and PROD levels were significantly increased only in the group C. In the adulthood, Vtg was significantly induced in the males in the group C. In generation F1, inhibition of CAT D activities and decrease of reproductive success were only found in pellet A group, but not in pellet B group. These findings indicate that the selection of diet is extremely important to assure veracity of the experiment results.