Microbial dechlorination of dioxins in estuarine enrichment cultures: effects of respiratory conditions and priming compound on community structure and dechlorination patterns

The effect of respiratory conditions and priming compound on dechlorination patterns of heptachlorodibenzo-p-dioxins (HpCDD) was investigated using estuarine sediment-eluted cultures in the presence and absence of 20 mM sulfate, and 0.2 microM 2-bromodibenzo-p-dioxin (2-BrDD) as a priming compound. Electron balance calculations based on fatty acid turnover, hydrogen production, and electron acceptor depletion/methane formation indicated that whereas fermentative processes dominated in sulfate-free incubations, sulfate-reduction was predominant in the sulfate-amended incubations. The dechlorination of 1,2,3,4,6,7,8-HpCDD exhibited the following trends: (i) the relative yields of product formation did not exceed 30% and the presence of 2-BrDD increased the yield by up to 10%; (ii) sulfidogenic conditions resulted in a lower 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) formation, and the presence of 2-BrDD decreased the formation of 2,3,7,8-TCDD by additional 4-5-fold; (iii) the presence of 2-BrDD effected a predominance in lateral (2,3,7,8 positions) over peri (1,4,6,9 positions)-dechlorination. Denaturing gradient gel electrophoresis (DGGE) banding patterns indicated significant shifts of microbial community structure in response to terminal electron accepting processes as well as to the presence of the priming compound. The latter resulted in a similar community structure independent of dioxin spike, indicating that subsets of populations in the sediment are capable of exploiting the new niche provided by the priming compound.     

缢蛏性腺和消化腺的生化组成与组化反应

福建省厦门海沧沿岸滩涂的缢蛏Sinonovacula constricta 的主要生殖期是10月,其两次高峰分别出现10月的上、下旬。缢蛏的性腺和消化腺发育过程中蛋白质、脂类、糖类与17种氨基酸的组成以及它们的组织化学反应的结果表明,在生殖周期中性腺与消化腺的蛋白质和脂类的变化大,而糖类都维持在低水平上,其最高量不超过9%干重。性腺和消化腺的蛋白贮存量随着性腺发育而增加,于成熟配子排放之前蛋白质量达到最大。排放之后蛋白质量和脂量皆下降,而糖量在性腺中却略有增加。在8～9月,消化腺和性腺内的脂量变化呈相反关系,表明当性腺形成过程它们之间可能有转化作用。性腺、消化腺的组化反应结果与它们的生化成分的分析相一致。性腺与消化腺的生化组成的相互关系表明,与脊椎动物相类似,缢蛏消化腺也有储存营养物质的作用。     

Esterification of vertebrate-like steroids in the eastern oyster (Crassostrea virginica)

The esterification of two model vertebrate steroid hormones - estradiol (E2) and dehidroepiandrosterone (DHEA) - was studied in the oyster Crassostrea virginica. The activity of acyl-CoA:steroid acyltransferase was characterized in microsomal fractions isolated from oyster digestive glands. The apparent Km and Vmax values changed with the fatty acid acyl-CoA used (C20:4, C18:2, C18:1, C16:1, C18:0 or C16:0), and were in the range of 9-17 microM, and 35-74 pmol/min/mg protein for E2, and in the range of 45-120 microM, and 30-182 pmol/min/mg protein for DHEA. Kinetic parameters were also assessed in gonadal tissue. The enzyme saturated at similar concentrations, although conjugation rates were lower than in digestive gland. Preliminary data shows that tributyltin (TBT) in the low microM range (1-50) strongly inhibits E2 and DHEA esterification, the esterification of E2 being more sensitive to inhibition than that of DHEA. Overall, results indicate that apolar conjugation occurs in oysters, in both digestive gland and gonads, at a very similar rate to mammals, suggesting that this is a well conserved conjugation pathway during evolution. Esterification, together with other mechanisms, can modulate endogenous steroid levels in C. virginica, and might be a target for endocrine disrupters, such as TBT.     

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.     

环沟格特蛤生化成分的周年变化

周年逐月采集环沟格特蛤（Ｋａｔｅｌｙｓｉａｒｉｍｕｌａｒｉｓ）,分别测定软体部、性腺、足肌和消化腺的蛋白质、脂肪、糖的含量,并测定性腺甾类激素和以细胞磷推算的ＤＮＡ和ＲＮＡ含量。结果表明,其蛋白质和脂肪含量的变化与性腺发育周期一致,糖的变化与蛋白质和脂肪相反,提示该种蛤在性腺生长周期糖类向蛋白质和脂肪转化,并且随着性腺的发育,消化腺和足肌的蛋白质和脂肪转运到性腺。环沟格特蛤的甾类激素、ＤＮＡ和ＲＮＡ的含量变化与蛋白质及脂肪的含量变化有一致性,提示甾类激素是促进性腺蛋白质合成的内因之一。     

Bioaccumulation of cadmium in the ascidian Styela clava (Herdman 1881)

The processes of bioaccumulation and elimination of cadmium by tissues of Styela clava (Herdman 1881) were investigated based on a semi-static two-compartment model. The kinetic parameters (accumulation rate constant k ₁, elimination rate constant k ₂, bioconcentration factor BCF, biological half-life t _1/2 and maximum equilibrium concentration C _Amax) were obtained by non-linear regression. The results showed that S. clava accumulates cadmium from its aquatic environment. BCF decreased with increasing metal concentration in the water, and when the accumulation achieved balance, C _Amax correlated positively with metal concentrations in the water. Concentrations of cadmium in different tissues of S. clava were: gonad > digestive gland ≈ other parts > tunic. The t _1/2 values for cadmium were 15.54-50.40 days in the accumulation process and 11.53-24.55 days in the elimination phase. The high rate of accumulation and elimination of cadmium from the body reveals the potential of the organism to be used as a biomonitor of short-term cadmium fluctuation in marine systems.     

Specific accumulation of organotin compounds in tissues of the rock shell,Thais clavigera

Concentrations of organotin compounds (butyltins and phenyltins) were determined in gonad, accessory sex organs, penis, digestive gland, kidney, radula with sac, oesophagus with crop, stomach, hypobranchial gland, rectum, mantle, osphradium, ctenidium, heart, salivary gland, head ganglia and muscle of imposex-exhibiting female and male rock shells (Thais clavigera), by gas chromatography with flame photometric detection (GC-FPD). Different tissue distributions were observed between butyltin and phenyltin compounds. More than 1000 ng TBT/g wet wt. were observed in ovary, digestive gland, kidney, heart, ctenidium, osphradium, stomach, head ganglia and penis, of both imposex-exhibiting females and males. More than 1000 ng TPT/g wet wt. were found in almost all tissues of both sexes. Approximately one-third or more of total TBT was accumulated in the digestive glands of both females and males, respectively. Meanwhile, approximately 40–50% and one-half of total TPT accumulated in the digestive glands of females and males, respectively.     

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.     

Significance of metallothioneins and lysosomes in cadmium toxicity and homeostasis in the digestive gland cells of mussels exposed to the metal in presence or absence of phenanthrene

Metallothioneins and lysosomes are known to be involved in cellular detoxication and sequestration of certain metals^1–3 and both have been identified in this role in elimination of copper from marine mussels (Mytilus edulis/galloprovincialis).³ Cadmium (Cd), however, has been shown to persist in the cells of the digestive gland for long periods with only minimal elimination. An experiment was designed to test the effects of Cd on the fragility of lysosomal membranes in the digestive cells as a measure of cellular injury,^4,5 metallothionein content of the digestive gland and cadmium concentration in this organ. Phenanthrene was used also to destabilise lysosomal membranes⁶ in order to test if increased lysosomal fragility interfered with cadmium metabolism and detoxication. The results demonstrated that Cd induced metallothionein synthesis and that elimination of Cd was minimal after 28 days in clear seawater. Lysosomal fragility was initially increased but this effect was soon reversed, even with continued exposure to Cd. The lysosomal destabiliser, phenanthrene, did not appear to affect accumulation of Cd or levels of metallothionein.     

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.     

大竹蛏同工酶组织特异性与多态性初步研究

用不连续垂直聚丙烯酰胺凝胶电泳对大竹蛏(Solen grandis Dunker)外套膜、鳃、性腺、消化腺、斧足5种组织6种同工酶(LDH、ADH、EST、MDH、ACP、SOD)进行组织特异性分析,并对吕四渔场海域大竹蛏自然种群生化遗传多样性进行了初步研究.结果表明:大竹蛏同工酶的活性及酶谱特征具有明显的组织特异性,6种酶在消化腺中的活性最强,LDH、EST、MDH和SOD酶谱具有明显的组织特异性;6种酶在消化腺和斧足中共检测到12个基因位点,其中Ldh-1、Ldh-2、Adh-1、Est-1、Mdh-1、Mdh-2、Mdh-3、Sod-2等8个基因位点为多态,多态位点比例为66.67%,平均每个位点的等位基因数N<,a>为2.5,有效等位基因数(N<,e>)为1.9,预期杂合度(H<,c>)为O.373 7,实际杂合度(H<,o>)为0.333 3,香农信息指数(I)为0.624 2.     

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.     

太平洋牡蛎内切葡聚糖酶基因的克隆、序列特性和表达分析

内切葡聚糖酶是一种重要的纤维素酶,在纤维素水解过程中起到重要作用。本实验利用RT-PCR和cDNA末端快速扩增技术(RACE)克隆获得了太平洋牡蛎(Crassostrea gigas)内切葡聚糖酶基因,并对其基因结构和系统进化进行了分析。内切葡聚糖酶cDNA全长802bp,开放阅读框架630bp,编码209个氨基酸。基因组DNA全长505 6bp,含有两段内含子长度分别为205 3和231 3bp。cDNA具有与已发现的内切葡聚糖酶基因相同的保守区结构和功能域,并且与其他已知贝类的内切葡聚糖酶有较高的同源性。利用RT-PCR方法研究了该基因在太平洋牡蛎消化腺、外套膜、鳃、肌肉、性腺和唇瓣组织中的表达,发现内切葡聚糖酶基因仅在消化腺中表达,而在其他组织中没有表达。     

Relationship between shell weight and cadmium content in whole digestive gland of the Japanese scallop Patinopecten yessoensis (Jay)

Seasonal and age-specific variations of cadmium (Cd) concentration in the digestive gland were investigated in the Japanese scallop Patinopecten yessoensis from Peter the Great Bay, Sea of Japan, with different degrees of Cd pollution. The seasonal changes in Cd concentrations of the digestive gland were inversely proportional to the dry weight of the gland. Concentrations of Cd and total Cd content (mug Cd per organ) increased with age (age-specific) to the same extent in contaminated and uncontaminated areas. There was also a strong positive correlation between Cd content in the whole digestive gland and shell weight and it is proposed that this relationship can be used as a new criterion for comparative evaluation of Cd levels in scallops from different areas We hypothesize that Cd is uptaken into scallops in proportion to the amount of calcium that absorbed through ion channels, and in addition, Cd in the digestive gland is in immobile forms (e.g. metal-rich granules) that accumulate with age. Moderate environmental pollution has no effect on the relationship between Cd content and shell size and the observed decrease in growth performance of the scallops from polluted areas may be due to other factors.     

Application of a 3-D chemical fate prediction model (FATE3D) to predict dioxin concentrations in the Tokyo Bay

A 3-D chemical fate prediction model (FATE3D) was applied to predict the dioxin concentrations in the seawater of Tokyo Bay, Japan. The simulations were carried out for a period of one year (from September 2002 to August 2003). Parameters such as meteorological data, flow field conditions, concentrations and sinking rates of organic particulate matter, initial and boundary conditions, and loading fluxes and physico-chemical properties of dioxins were used as the model inputs.The simulation results compared favorably with the field measurements of dioxin concentrations in the bay for both the particulate and dissolved phases, indicating the validity and predictive capability of the model. Furthermore, the differences in the seasonal cycles and distributions between the particulate- and dissolved-phase dioxins in the bay were estimated from the simulation results.However, the particulate-phase dioxin concentrations in the bottom layers (+1 m from the bottom) were underestimated, probably because the resuspension process was not taken into account in the model. The improvement of the model's predictive capability, including the resuspension process, shall be the focus of our next study.     

3种海洋养殖贝类体内的脂肪酸组成

分析了华贵栉孔扇贝Ｃｈｌａｍｙｓｎｏｂｉｌｉｓ（Ｒｅｅｖｅ）、合浦珠母贝Ｐｉｎｃｔａｄａｍａｔｅｎｓｉｉ（Ｄｕｕｋｅｒ）、翡翠贻贝Ｐｅｒｎａｖｉｒｌｄｉｓ（Ｌｉｎｎａｅｕｓ）的软体部、肝胰腺和生殖腺的脂肪酸组成及含量。３种贝的各组织中,不饱和脂肪酸含量均较饱和脂肪酸含量高。多不饱和脂肪酸（ＰＵＦＡ）含量丰富,其中二十碳五烯酸（ＥＰＡ）和二十二碳六烯酸（ＤＨＡ）含量相当高,占总脂肪的２５．２６％－２７．２１％。３种贝中,翡翠贻贝的油含量最高（１３．６８％）,软体部的ＥＰＡ和ＤＨＡ含量也较高（２６．９５％）,最具开发利用价值。     

Bleached kraft pulpmill effluents and TCDD: Effects on biotransformation and testosterone status of mature male rainbow trout (Salmo gairdneri)

The aim of this study was to examine the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, given intraperitoneally) alone and together with bleached kraft pulpmill effluents (BKME) on fish biotransformation reactions and testosterone levels in mature male rainbow trout (Salmo gairdneri Richardson). The results show extremely high induction in liver and kidney monooxygenase (PSMO) activities in 3 weeks caused by TCDD alone or TCDD together with BKME. BKME alone did not cause significant effects. 7-Pentoxyresorufin O-dealkylase activity, previously shown to be an indicator of phenobarbital-type induction, was shown to be highly inducible by TCDD in fish. Plasma testosterone levels, although decreased in another subacute exposure to BKME, showed only slight changes due to TCDD and TCDD + BKME.     

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.     

Role of metallothioneins in Cu and Cd accumulation and elimination in the g gill and digestive gland cells of mytilus galloprovincialis lam.

The data presented in this paper demonstrate that exposure of mussels to Cu (40 μg/liter) or Cd (200 μg/liter) for a period of 2 days is sufficient to induce Cu or Cd thioneins in the gills of the metal-exposed animals. The concentrations of Cu and Cd thioneins increase during the period of metal exposure (3–4 weeks), confirming the fact that metallothioneins play a fundamental role in the accumulation of these metals in the tissues examined (gills and digestive gland). Moreover, it has been demonstrated that when metal-loaded mussels are returned to the field for the recovery period, Cu is rapidly eliminated from the gill and digestive gland cells, showing a biological half-life of 9–10 days, whereas Cd is released much more slowly from the tissues, only about 50% of the total metal being lost after 4 months of detoxification. Interestingly enough, during the detoxification period the concentration of the metal bound to thioneins also follows the same pattern as the total metal present in the tissues, in fact the concentration of Cu thioneins decreases near the control level in about 24 days but, in contrast, the Cd-thionein concentration in the cytisol of the gill and digestive gland cells remaains high, decreasing only by about 40% at the end of the 4 month recovery period.The data presented also demonstrate that when Cd-loaded, detoxified mussels are exposed to Cu (100 μg/liter) for 6 h, the metal taken up by the gill cells is able to displace the Zn which is always present in the Cd-thionein fraction, but it does not, however, displace the Cd present. In this case also, period (20 days), whereas the Cd-thionein content decreases slightly. Such data seem to indicate that the metabolic characteristics of thioneins are closely related to the metal bound to them, particularly where the elimination rate of the metals from the cells is concerned.