Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

Seasonal variation of histopathological and histochemical markers of PAH exposure in blue mussel (Mytilus edulis L.)

The aim of this work was to study seasonal variation of histopathological and histochemical markers in blue mussels (Mytilus edulis L.) exposed to pyrogenic PAH contaminants. Mussels were collected in January, June, September and October from a sampling site in the vicinity of the discharge from an aluminium smelter and from a clean reference site. Histopathological analysis was carried out on the digestive gland (DG) and the gonads, lipofuscin and neutral lipids were analysed in the DG. Clear responses in lipofuscin and neutral lipids were detected in the DG of mussels collected from the polluted site at some sampling times. Moreover, these mussels presented atrophy in digestive tubules and haemocytic aggregates in the gonad and DG. However, in all parameters studied, the magnitude of the response showed clear seasonal variation.     

Effect of oil pollution on gluthione and relative enzyme in oyster(Saccostrea cuculiata)

The oysters ( Saccostrea cuculiata ) were collected from four stations around Xiamen Island (Lundu Port, Xinglin Bay, Tong" an Bay, Huangcuo). The relation between the level of petroleum hydrocarbon in whole tissue and the contents of glutathione (GSH), the activity of selenium-dependent glutathione peroxidase (Se-GPx) and glutathione S-transferase (GST) in digestive gland and gill were investigated. The results showed: (1) The contents of petroleum hydrocarbon in oyster collected from four stations (Lundu Port, Xinglin Bay, Tong'an Bay, Huangcuo) were 380.68, 112.34, 27.31, 20.37μg/g wet weight, respectively; (2) the activity of Se-GPx and GST in digestive gland was lower than that in gill, and the content of GSH seemed reversibly; (3) among the four stations, both Se-GPx and GST activity of digestive gland and gill in Saccostrea cuculiata sampled from the four stations showed a good correlation with whole tissue petroleum hydrocarbon, could be as biomarkers of sea oil pollution.     

The fine structure of lysosomal membranes and endoplasmic reticulum in the digestive cells of Mytilus edulis exposed to anthracene and phenanthrene

The stability of lysosomes in the digestive cells of Mytilus edulis is affected by changes in both the chemical and physical conditions of the environment.¹ This type of response must reflect, at least in part, some change in the organisation of the lysosomal membrane, which is a structure which can be resolved by the electron microscope. However, conventional methods of tissue preparation have produced low contrast images of the digestive gland and damage to the lysosomal membranes. Recently a cryopreparation technique² has produced lysosomal membrane preparations in the digestive cells of the digestive gland of Mytilus which have high contrast and structural integrity (Fig. 1) in control animals. This method has been used to demonstrate pathological alterations in the lysosomal membrane induced by the polynuclear aromatic hydrocarbon (PNAH) phenanthrene. Anthracene, an isomeric PNAH, had no effect on the morphology of the lysosomal membrane. Phenanthrene also induced apparent proliferation of smooth endoplasmic reticulum in the digestive cells.     

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

DNA adducts in gills and digestive gland, as well as polycyclic aromatic hydrocarbon (PAH) tissue levels were analysed in blue mussels (Mytilus spp.) from Nordic coastal areas (Iceland, Norway and Sweden) with diffuse or point sources of PAHs of various origins. Both DNA adduct and PAH tissue levels were generally low, indicating low PAH exposure to the mussels in the areas studied. DNA adducts were found to be higher in gills than in digestive gland of the mussels at all sites studied. Elevated DNA adduct levels in gills were found at 6 sites out of 18 compared to reference sites in respective coastal zones. Adduct levels ranged from 0.5 to 10 nmol adducts/mol normal nucleotides, being highest in mussels from Reykjavík harbour, Iceland (intertidal mussels), and from Fiskaatangen, Norway (subtidal mussels). Total PAH tissue levels in the mussels ranged between 40 and 11,670 ng/g dry wt., and were significantly correlated with DNA adduct levels (r(2)=0.73, p<0.001). PAH ratio values indicated that the PAHs were in most cases of pyrolytic origin, but with petrogenic input near harbours and an oil refinery.     

A multi-biomarker approach to assess effects of Triclosan in the clam Ruditapes philippinarum

The effects of 7 days’ exposure to differing Triclosan (TCS) concentrations (300, 600, and 900 ng/L) were investigated in the clam Ruditapes philippinarum. Vitellogenin (Vg)-like protein levels in haemolymph and digestive gland from males and females, gill acetylcholinesterase (AChE) activity, superoxide dismutase (SOD) and catalase (CAT) activities in gills and digestive gland, and gill lipid peroxidation (LPO) were measured. The highest TCS concentrations decreased significantly Vg levels in male haemolymph and digestive gland, whereas no significant variations were found in females. The highest TCS concentrations increased significantly SOD activity in gills, but decreased it in digestive gland. No changes in CAT activity were observed. In gills, TCS reduced significantly AChE activity, but it did not induce significant variations in LPO. Our study demonstrates that TCS alters biochemical parameters in R. philippinarum, even at environmentally realistic concentrations, and suggests differing modes of action of the contaminant, in clams at least.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.     

文蛤一种球形病毒的分离纯化及细胞病理变化观察

为研究一种球形病毒引起的文蛤(Meretrix meretrix)细胞病理变化及该病毒的分类地位,作者运用透射电镜对异常死亡文蛤的细胞病理变化及感染的病毒粒子进行了超微结构观察,并通过差速离心、酒石酸钾超速离心和氯化铯密度梯度离心结合等方法对首次对该病毒粒子进行了分离纯化。结果表明:患病个体的鳃、消化盲囊、消化道和外套膜均大量存在一种病毒颗粒,该病毒呈球形,无囊膜,直径45~55 nm,核心与核衣壳间无明显界限,未发现病毒包涵体,该病毒存在于细胞间质及被破坏的细胞质中。该病毒粒子的感染引起的细胞病理变化主要表现为:细胞核染色质集聚,溶酶体增多,线粒体肿涨,粗面内质网膨胀,肌原纤维排列混乱,细胞结构变得无规则直至解体。病毒粗提液于4℃150 000 g氯化铯密度梯度离心20 h可该病毒粒子提纯,经纯化后负染显示,该病毒粒子呈球形,二十面体对称,无囊膜,直径在60~75 nm,表面结构粗糙,壳粒结构较明显,该病毒粒子在氯化铯中的浮密度为1.554~1.582 g/m L。本文结果为文蛤死亡病原的研究提供了基础资料。     

原油污染对栉孔扇贝抗氧化酶活性的影响

以原油水溶性成分(water soluble fraction of crude oil,WSF)为污染物,采用暴露实验法,研究了栉孔扇贝(Chlamys farreri)鳃和消化腺组织中超氧化物歧化酶(Superoxide Dismutase,SOD)和过氧化氢酶(Catalase,CAT)活性的变化.结果表明,WSF污染下,鳃和消化腺组织SOD和CAT酶活性随暴露时间增加一般表现为降低-升高-降低的趋势,酶活性达到最高的时间随浓度不同而变化.第1天时消化腺SOD在0.08 mg/L浓度下被诱导,而后随时间增加表现为抑制效应;浓度为0.21和0.88 mg/L时消化腺SOD酶活性被抑制,随暴露时间延长而活性增加.暴露时间为4d时,石油烃浓度在0.08和0.88 mg/L时鳃组织SOD酶活性均被抑制,而浓度为0.21 mg/L时被诱导.消化腺和鳃组织SOD可以作为扇贝被污染胁迫的指标.     

Biomarker responses in Macoma nasuta (Bivalvia) exposed to sediments from northern San Francisco Bay

Our study investigates biomarker responses and survival of Macoma nasuta exposed to sediments collected from six locations in northern San Francisco Bay. Biomarkers analyzed were stress proteins (hsp70) in gill, mantle and digestive gland, lysosomal membrane damage and histopathologic lesions. Sediments and clam tissues were analyzed for a comprehensive suite of heavy metals and trace organic pollutants. Sediment grain size and organic carbon content were determined. Clams accumulated metals, polyaromatic hydrocarbons (PAHs) and organochlorine pesticides (aldrin and p,p(')-DDT and its metabolites p,p(')-DDD and p,p(')-DDE). Pearson and Spearman correlation analysis revealed that mortality, hsp70 in gill and histopathologic lesion scores in gonads, and lysosomal membrane damage were significantly correlated with tissue concentrations of DDT and/or its metabolites. Tissue concentrations of metals, in particular nickel, chromium, and copper, were associated with macrophage aggregates in digestive gland and germ cell necrosis. Cadmium was linked to mortality and lysosomal membrane damage.     

Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations

The high concentrations of cadmium recorded in the digestive gland of cephalopods from various temperate and subpolar waters suggest that these molluscs have developed efficient cadmium detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland cells was investigated in seven cephalopod species from the Bay of Biscay (France) and the Faroe Islands. In most species, cadmium was mainly found in the cytosolic fraction of the digestive gland cells, reaching up to 86% of the total cadmium for the squid Loligo vulgaris from the Bay of Biscay. But species with the highest total level of cadmium showed a higher percentage of cadmium associated to insoluble compounds. The quantification of metallothioneins (MTs) by the polarographic method was performed in order to evaluate the involvement of these proteins in the detoxification of the high amounts of bioaccumulated cadmium. Metallothionein levels in cephalopods ranged form 742 +/- 270 to 3478 +/- 1572 microg/g wet weight. No relationship could be established between total cadmium, cytosolic cadmium and MT levels suggesting the occurrence of other Cd-binding ligands. Although these proteins have not been characterised, as cadmium in the digestive gland of cephalopods is mainly associated with soluble ligands, a high potential transfer to predators can be predicted.     

Variability of heat shock proteins and glutathione S-transferase in gill and digestive gland of blue mussel,Mytilus edulis

Glutathione S-transferase (GST) and heat shock proteins (hsps) 40, 60, 70 and 90 were determined by immunoblotting using actin as an internal control in Mytilus edulis from one station outside (site1) and three stations within (sites 2-4) Cork Harbour, Ireland. Comparisons were made between gill and digestive gland and between sites. Gill shows generally higher hsp 60, 70 and 90 while digestive gland has higher hsp 40. Site 1 showed higher gill hsps 40 and 70 than sites 2-4 while gill GST was higher in sites 3 and 4 than 1 and 2. Comparison with sites in the North Sea (site 5: outside Tj?rn? in The Koster archipelago in the Skagerack) and Baltic Sea (site 6: Ask? island) also revealed lower hsps 40 and 70 in site 6 (low salinity) than site 5 (high salinity) although hsps 60, 70 and 90 were detectable in digestive gland unlike sites 1-4. Previously, only hsp 70 had been studied at these sites [Mar. Environ. Res. 39. (1995), 181]. At the mRNA level, gill hsp 70 is 80-fold higher at Tj?rn? than Ask?. These data suggest that, while salinity may slightly decrease hsp 40 and 70, both hsp 70 and GST are selectively up-regulated by approx. 10- and 3-fold, respectively, at Tj?rn? compared to the other sites which we attribute to exposure to more widely fluctuating pollution levels.     

The development of a physiologically-based pharmacokinetic model using the distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the tissues of the eastern oyster (Crassostrea virginica)

A physiologically-based pharmacokinetic model (PBPK) was developed to describe the kinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the eastern oyster (Crassostrea virginica). The estimated t(1/2) of elimination for a bolus dose of TCDD in C. virginica is approximately 14-24 days based on both the experimental data and the PBPK model. The highest dioxin concentration reached during 28-days was in the digestive gland followed by the mantle, gonad, hemolymph, gill, adductor muscle, and the kidney/heart. A binding protein for 2,3,7,8-TCDD had been reported in the literature for both the digestive gland and gonad. Incorporating a binding component in the model resulted in a better fit for the data. The PBPK model predicted the distribution and the elimination concentrations for 2,3,7,8-TCDD within each of the tissue compartments. This model will serve as a useful tool for predicting the kinetics of other persistent organic pollutants as well as, allow for a more refined ecological risk assessment by estimating dioxin concentrations in sensitive tissues such as the gonad.     

Evaluation of neutral red retention assay,micronucleus test,acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring

The neutral red lysosomal retention assay (NRR) of the haemocytes, and the acetylcholinesterase activity (AChE) in the haemolymph, the digestive gland, the gills and the mantle/gonad complex have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (northern Greece) in June and October 2001. The validity of performing the above core biomarkers is supported, firstly by their ability to respond to different pollution levels and, secondly, by the significant linear correlation among them. The evaluation of the micronuclei frequency (MN) has been performed in gill tissue and haemocytes of the same mussels and, according to the results, it needs more research in order its use as stress indices to be validated. In addition, the first results on cAMP levels in the gills, the mantle/gonad complex and the digestive gland, whose concentrations correlated to both, NRR and AChE introduce this signal transduction molecule as a new, promising biomarker.     

A description of chloride cell and kidney tubule alterations in the flatfish Solea senegalensis exposed to moderately contaminated sediments from the Sado estuary (Portugal)

The effects of sediment-bound contaminants on kidney and gill chloride cells were surveyed in juvenile Solea senegalensis exposed to fresh sediments collected from three distinct sites of the Sado Estuary (Portugal) in a 28-day laboratorial assay. Sediments were analyzed for metallic contaminants, polycyclic aromatic hydrocarbons and organochlorines as well as for total organic matter, redox potential and fine fraction. The potential for causing adverse biological effects of each surveyed sediment was assessed by comparison of contaminant levels to available guidelines for coastal sediments, namely the Threshold Effects Level (TEL) and the Probable Effects Level (PEL). The Sediment Quality Guideline Quotient indices (SQGQ) were calculated to compare the overall contamination levels of the three stations. A qualitative approach was employed to analyze the histo/cytopathological traits in gill chloride cells and body kidney of fish exposed to each tested sediment for 0, 14 and 28 days. The results showed that sediment contamination can be considered low to moderate and that the least contaminated sediment (from a reference site, with the lowest SQGQ) caused lesser changes in the surveyed organs. However, the most contaminated sediment (by both metallic and organic xenobiotics, with highest SQGQ) was neither responsible for the highest mortality nor for the most pronounced lesions. Exposure to the sediment presenting an intermediate SQGQ, essentially contaminated by organic compounds, caused the highest mortality (48%) and the most severe damage to kidneys, up to full renal necrosis. Chloride cell alterations were similar in fish exposed to the two most contaminated sediments and consisted of a pronounced cellular hypertrophy, likely involving fluid retention and loss of mitochondria. It can be concluded that sediment contamination considered to be low or moderate may be responsible for severe injury to cells and parenchyma involved in the maintenance of osmotic balance, contributing for the high mortality levels observed. The results suggest that sediment-bound organic contaminants such as PAHs (polycyclic aromatic hydrocarbons) and PCBs (polychlorinated biphenyls) may be very toxic to the analyzed organs, especially the kidney, even when present in low-risk concentrations.     

A description of chloride cell and kidney tubule alterations in the flatfish Solea senegalensis exposed to moderately contaminated sediments from the Sado estuary (Portugal)

The effects of sediment-bound contaminants on kidney and gill chloride cells were surveyed in juvenile Solea senegalensis exposed to fresh sediments collected from three distinct sites of the Sado Estuary (Portugal) in a 28-day laboratorial assay. Sediments were analyzed for metallic contaminants, polycyclic aromatic hydrocarbons and organochlorines as well as for total organic matter, redox potential and fine fraction. The potential for causing adverse biological effects of each surveyed sediment was assessed by comparison of contaminant levels to available guidelines for coastal sediments, namely the Threshold Effects Level (TEL) and the Probable Effects Level (PEL). The Sediment Quality Guideline Quotient indices (SQGQ) were calculated to compare the overall contamination levels of the three stations. A qualitative approach was employed to analyze the histo/cytopathological traits in gill chloride cells and body kidney of fish exposed to each tested sediment for 0, 14 and 28 days. The results showed that sediment contamination can be considered low to moderate and that the least contaminated sediment (from a reference site, with the lowest SQGQ) caused lesser changes in the surveyed organs. However, the most contaminated sediment (by both metallic and organic xenobiotics, with highest SQGQ) was neither responsible for the highest mortality nor for the most pronounced lesions. Exposure to the sediment presenting an intermediate SQGQ, essentially contaminated by organic compounds, caused the highest mortality (48%) and the most severe damage to kidneys, up to full renal necrosis. Chloride cell alterations were similar in fish exposed to the two most contaminated sediments and consisted of a pronounced cellular hypertrophy, likely involving fluid retention and loss of mitochondria. It can be concluded that sediment contamination considered to be low or moderate may be responsible for severe injury to cells and parenchyma involved in the maintenance of osmotic balance, contributing for the high mortality levels observed. The results suggest that sediment-bound organic contaminants such as PAHs (polycyclic aromatic hydrocarbons) and PCBs (polychlorinated biphenyls) may be very toxic to the analyzed organs, especially the kidney, even when present in low-risk concentrations.     

皱纹盘鲍主要器官的亚显微结构

对皱纹盘鲍的胃、直肠、消化腺、肾、鳃、心脏、外套膜、足等器官的亚显微结构进行了研究。结果表明 :(1 )各消化器官的组织结构中富含酶原颗粒和分泌颗粒 ,这些颗粒具有消化和抗病的功能 ;(2 )鳃和心肌的组织结构中富含线粒体 ,这与其活动量大密切相关 ;(3 )外套膜的组织结构中富含分泌颗粒 ,这些颗粒物质参与贝壳的形成和体液免疫 ;(4)肾实质中静脉呈网状分布 ,这与体液的有效过滤有关 ;(5 )腹足的肌纤维成束 ,并形成三维的网状结构 ,以适应匍匐与吸附的生活方式     

Cadmium accumulation by the blue crab, callinectes sapidus: Involvement of hemocyanin and characterization of cadmium-binding proteins

The accumulation of cadmium from seawater by the blue crab, Callinectes sapidus, was studied as a function of metal concentration and exposure time, with special emphasis on cadmium-binding proteins. Cadmium was found, in decreasing order of magnitude, in gills, digestive gland and hemolymph. When exposed to 0·5 ppm cadmium for 2–24h, virtually all of the cadmium in the cytosolic fraction of the gill was associated with a low molecular weight (LMW) cadmium-binding protein (MW 8000). However, after 48h of exposure only 50% of the cadmium in the cytosol was bound to this protein. The rest wasfound to be associated with proteins of a molecular weight of 300 000 and 60 000. This pattern of cadmium distribution did not change over a 12-day depuration period. Similar results were obtained upon exposure to 0·1 ppm cadmium. The pattern of cadmium accumulation in the cytosolic fraction of the digestive gland was in marked contrast to that observed for the gill. Initially, the cadmium was distributed over three low molecular weight fractions. During depuration the distribution of cadmium changed and all of the metal became bound to a low molecular weight protein (MW 9000). The cadmium concentrations in the gill and digestive gland remained essentially constant during depuration (12 days). The LMW cadmium-binding proteins were purified by a combination of gelpermeation and ion-exchange chromatography. Their molecular weight, spectral properties and amino acid composition are characteristic of the vertebrate metallothioneins. During exposure to cadmium the metal rapidly appeared in the hemolymph, mainly associated with hemocyanin. During depuration cadmium was transferred from the hemolymph to the digestive gland, demonstrating that hemocyanin acts as a carrier in trace metal transport.     

Ubiquitination and carbonylation as markers of oxidative-stress in Ruditapes decussatus.

Environmental pollutants, such as metals, are widespread in aquatic environments and can lead to the formation of reactive oxygen species (ROS). ROS are highly toxic in marine species since they can cause serious reversible and irreversible changes in proteins including ubiquitination and modifications such as carbonylation. This study aimed to confirm the potential of ubiquitination and carbonylation as markers of oxidative stress in the clam Ruditapes decussatus (Veneroida, Veneridae) exposed to cadmium (40 microg/L). After 21 days of exposure clams were dissected into gills and digestive gland. Cytosolic proteins of both tissues were separated by two-dimensional electrophoresis (2-D SDS-PAGE) and analysed by immunobloting. Higher ubiquitination and carbonylation levels were in digestive gland of contaminated organisms. These results confirm the potential of ubiquitination and carbonylation as a sensitive and specific marker of oxidative stress in marine bivalves. In this approach, changes in protein structure provide options for affinity selection of sub-proteomes for 2D SDS-PAGE, simplifying the detection of protein biomarkers using proteomic approach.