Effects on UDP glucuronosyl transferase, glutathione transferase, DT-diaphorase and glutathione reductase activities in rainbow trout liver after long-term exposure to PCB

In the present study, long-term exposure to PCB resulted in marked induction of the xenobiotic biotransformation enzymes UDP glucuronosyl transferase, glutathione transferase and DT-diaphorase (measured as menadione reductase and DCPIP reductase) in rainbow trout 15 or 20 weeks post-injection during the experimental period. The increase of the phase II enzymes were paralleled by a marked increase of the glutathione reductase activity. These results indicate that prolonged exposure to PCB results in increased production of organo-radicals.     

Gold nanoparticles and oxidative stress in Mytilus edulis

Little is known about potential environmental impact of nanoparticles. Gold nanoparticles can cause unexpected biological responses. Here, Mytilus edulis were exposed (24h) to gold-citrate nanoparticles (GNP), menadione and both compounds simultaneously (GNP/menadione). Protein ubiquitination and carbonylation were determined in gill, mantle and digestive gland, along with traditional oxidative stress biomarkers; catalase activity and neutral red retention time assay (haemolymph). 2DE gels were performed on gill proteins (menadione; GNP/menadione). Our results reveal that GNP may induce oxidative stress.     

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: glutathione reductase as a target

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.     

Effects of cadmium, zinc and nitrogen status on non-protein thiols in the macroalgae Enteromorpha spp. from the Scheldt Estuary (SW Netherlands, Belgium) and Thermaikos Gulf (N Aegean Sea, Greece)

Enteromorpha prolifera (Scheldt Estuary) and E. linza (Thermaikos Gulf) were incubated at three salinities with 100 and 200microgL(-1)Cd and Zn. The objective was to measure effects of Cd, Zn and nitrogen (N) status on the pools of metal-binding non-protein thiols: glutathione and phytochelatins, (gamma-glutamyl-cysteinyl)(n)-glycine (PC). In E. linza, ammonium pools were higher, but amino acid pools, total N and protein contents were lower than in E. prolifera. Reduced glutathione (GSH) pools were positively correlated with free glutamate and protein contents. In E. linza GSH pools increased and the ratio of reduced to oxidized glutathione (GSH:(GSH+0.5GSSG)), an indicator of oxidative stress, decreased with Cd contents, indicating Cd-induced glutathione oxidation. Total glutathione pools (reduced plus oxidized) ranged from 16nmolSgdwt(-1) in controls (at 0.5micromolCdgdwt(-1)) to 179nmolSgdwt(-1) (at 1.9micromolCdgdwt(-1)) at the highest cadmium dosage. Cadmium stimulated PC synthesis in E. prolifera which suggests that in N-rich algae, glutathione pools were high enough for PC synthesis. In both species GSH and protein increased with Zn contents, whereas GSH:(GSH+0.5GSSG) decreased, which would indicate Zn-induced oxidative stress; in E. linza, at the highest salinity the glutathione redox ratio decreased from 0.61 (at 2.9micromolZngdwt(-1)) to 0.26 (at 4.9nmolSgdwt(-1)) (at 0.5molCdgdwt(-1)). PCs were not synthesized in response to Zn, which may have resulted in Zn-induced GSH oxidation. The presence of both oxidative effects (Cd, Zn) and detoxification (Cd) could be identified by observing the responses of glutathione and PC pools to metal stress.     

Levels of cellular glutathione and metallothionein affect the toxicity of oxidative stressors in an established carp cell line.

The comparative toxicity of a variety of oxidative stressors was studied in the epithelioma papulosum cyprini line from carp using the neutral red cytotoxicity assay. LC50's decreased in the order t-butylhydroperoxide > hydrogen peroxide > diquat > paraquat. The cytotoxicity of hydrogen peroxide was significantly reduced when the cells were grown in L-15 medium rather than MEM and this could be attributed to elevated cellular glutathione and metallothionein levels and higher activities of GSH-dependent detoxification systems. The protective effect of metallothionein in radical scavenging was demonstrated by decreased toxicity of the redox-cycling toxicants, diquat and menadione after metallothionein levels had been pre-induced by Cd-exposure. This study demonstrates the relationship between toxic effects of oxidative stressors and expression of detoxification systems in fish.     

金鼓鱼幼鱼对蛋白质的适宜需要量研究

为确定金鼓鱼(Scatophagus argus)日粮中蛋白质的适宜添加量,作者以酪蛋白为蛋白源、鱼油为脂肪源,配制脂肪水平为8%,蛋白水平分别为24%、28%、32%、36%、40%的5种配合饲料(D1~D5),在海上网箱中以初始体质量为4.3 g左右的幼鱼开展养殖实验。8周后,测定实验鱼增质量率、特定生长率、蛋白质效率、饲料系数、消化酶和一些免疫及抗氧化指标。结果显示,随着饲料蛋白水平的升高,金鼓鱼的增质量率、特定生长率、蛋白质效率等均呈现先上升后下降的趋势,以D3组最高,3项指标均显著高于D1、D2组;饵料系数呈现先下降再上升趋势,以D3组最低,且显著低于D1、D2组。饲料蛋白水平也显著影响金鼓鱼的相关生理生化指标,其中D3和D4组的全鱼蛋白含量、消化道蛋白酶活性和一些免疫抗氧化指标最高,全鱼脂肪含量最低。根据饲料蛋白含量与增质量率的线性回归分析,确定金鼓鱼幼鱼饲料中的适宜蛋白水平为35%。     

An improved electrochemical method for the quantification of metallothioneins in marine organisms

A six-to nine-fold increase in detection limits of the Brdic?ka polarographic assay for metallothioneins has been achieved through the employment of a commercially available static mercury drop electrode (SMDE) and reduced temperatures. At 16°C detectable concentrations of metallothionein (MT-1) from the crab Scylla serrata were in the range of 5 to 10 μg litre^-1. Operating conditions and electrode preparation are discussed. For a number of thiolic molecules a linear relationship between polarographic response and either the number of cysteinyl residues or the molecular weight was noted. In the latter case on enhancement of response by sulfur-metal bonds is suggested. The utility of this assay permits a rapid quantitative assay for these proteins at basal levels in marine invertebrates and other organisms.     

Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1). Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.     

Determinants of species susceptibility to oxidative stress: A comparison of channel catfish and brown bullhead

Xenobiotic-mediated productions of reactive oxygen species, via enzymemediated redox cycling, have been implicated in a variety of toxicological phenomena including lipid peroxidation, enzyme inactivation and oxidative DNA damage leading to cancer. A comparison was undertaken of two benthic freshwater fish species that appear to differ markedly in their susceptibility to chemical carcinogenesis—the channel catfish (Ictalurus punctatus) and the more cancer-prone brown bullhead (Ameriurus nebulosus)-in terms of basic biochemical characteristics related to oxidative stress. This has included analysis of microsomal redox cycling of model xenobiotics (e.g. menadione) as well as antioxidant and other detoxifying enzymes in hepatic tissue of the two species. In addition, endpoints of oxidative stress, such as altered glutathione status and oxidative DNA damage, were examined. These studies have revealed numerous qualitative and quantitative differences between the two species both in terms of basal enzyme activities and in species response to model prooxidants. For example, bullhead appear to possess a greater capacity for microsomal redox cycling of xenobiotics, but have glutathione-dependent defense systems less able to withstand oxidative challenge. These and other interspecific differences have allowed for an improved understanding of the basic mechanisms which may underly species susceptibility to oxidative stress and critical manifestations such as cancer.     

The simultaneous immunological detection of four stress-70 protein isoforms in Mytilus edulis

Organisms exposed to adverse environmental conditions exhibit a stress-70 protein response which is highly conserved from bacteria to man. Immunological analysis of the stress-70 response in mussels is of interest for its potential use in marine biomonitoring. We have analysed the gill tissues of Mytilus edulis individuals for changes in stress-70 complement after exposure to 48 h of temperature stress. Using a commercially available monoclonal antibody raised against human hsp70 and epitope mapped to a conserved ATP binding region, the authors were able to detect by western analysis constitutively-expressed 72kDa and 78kDa isoforms in control animals acclimated at normal temperatures. After 48 h of heat stress, induction of a 70kDa protein was clearly seen along with an increase in the level of the 72kDa protein. Western analysis using two-dimensional gel electrophoresis indicated that the antibody recognised four stress-70 isoforms (70, 72, 72 and 78kDa). This simultaneous detection of four members of the stress-70 family will permit studies on the differential regulation of each of the four members of the multigene family in response to a variety of environmental Stressors.     

Neuroendocrine disruption in Mya arenaria clams during gametogenesis at sites under pollution stress

This study examined the neuroendocrine status of clams on intertidal mud flats in the St. Lawrence Estuary and Saguenay Fjord areas during late gametogenesis. The impact of pollution was determined by a test battery of early stress markers (metallothioneins, heme levels, glutathione S-transferase activity), tissue damage (lipid peroxidation and DNA damage) and morphologic characteristics (age, soft-tissue weight ratio and growth index). Neuroendocrinal status was examined by tracking serotonin and dopamine metabolism, monoamine adenylate cyclase activity in synaptosomes, monoamine oxidase and arachidonate cyclooxygenase activities in relation to gametogenetic activity: pyrimidine synthesis, (aspartate transcarbamoylase activity or ATC), vitellogenin-like proteins and gonado-somatic index. The results show that clam soft tissue weights were reduced at sites close to harbours and higher at sites near domestic wastewater outfalls. The age-to-length ratio of clams was generally higher at impacted sites, suggesting reduced growth. The biomarkers of stress or damage confirmed that oxidative stress, DNA damage, metallothioneins and glutathione S-transferase activity were significantly increased at varying degrees, at the polluted sites. Vitellogenin-like proteins and gametogenetic activity were significantly increased at sites influenced by domestic wastewaters. Furthermore, the clams were still in active gametogenesis and not ready for spawning, as indicated by the concordance of the serotonin/dopamine ratio with vitellogenin-like proteins and pyrimidine synthesis. However, gonadal cyclooxygenase activity was increased at polluted sites and significantly correlated with most of the stress biomarkers, suggesting that the clams were in a state of inflammation rather than at the spawning stage. Finally, a multivariate analysis revealed that the sites were readily discriminated with high efficiency (>71%) and that both neuroendocrine physiological markers and stress responses were identified as the major components, thus explaining the global physiological response of the clams. We conclude that the effects of pollution compromise the clams' health status and that the initiation of gametogenesis in environments contaminated by municipal wastewaters or harbours contributes to the toxic effects of pollution.     

Changes in iron concentrations and bio-availability during an open-ocean mesoscale iron enrichment in the western subarctic Pacific,SEEDS II

A patch of water in the western subarctic gyre (low iron concentration, <0.02 nM) was fertilized twice with 322 and 159 kg of iron to induce a phytoplankton bloom. In order to understand the changes in iron distribution and bio-availability throughout the evolution and termination phase of the iron-induced bloom, iron concentrations were measured at stations inside and outside of the iron-fertilized patch, and shipboard culture experiments using iron and desferrioxamine B (DFB) inoculation to regulate iron availability were conducted 5 times with water collected from the center of the iron-fertilized patch on D2, D7, D11, D17 and D23.After the iron fertilization, we observed a significant increase in dissolved iron (1.38 nM at 5 m depth) at the center of the patch (D1). Dissolved iron concentrations subsequently decreased to an ambient level (~0.08 nM) on D16–D17, despite the second iron fertilization made on D6. During the 4-day incubations of the shipboard culture experiments, excess DFB-inoculated treatment inhibited the phytoplankton growth compared to the controls for D2, D7 and D11 patch water. This indicated that available iron existed in the iron-fertilized patch at least until D11. Moreover, iron-inoculated treatments induced growth of large-sized phytoplankton with an accompanying silicate decrease for D7, D11 and D17 patch water, but not for D23 patch water. These results indicated that large diatoms, which can respond to additional iron inoculation, existed in the iron-fertilized patch in evolution and early termination phase of the iron-induced bloom (at least until D17); however, there was no significant amount of large diatoms, which could rapidly respond to iron, in late termination phase (D23) of the iron-induced phytoplankton bloom.     

花生四烯酸调控网络在贻贝麻痹性贝毒代谢中的作用基于转录组和代谢组联合分析

紫贻贝（Mytilus galloprovincialis）是富集麻痹性贝类毒素（Paralytic Shellfish Toxins, PSTs）能力很强的代表性双壳贝类,但研究表明紫贻贝暴露于PSTs也会引起机体炎症反应,其作用机理及对毒素代谢影响尚不清楚。本研究采用转录组学与代谢组学联合分析技术,比较了产PSTs的链状亚历山大藻（Alexandrium catenella）暴发的不同时期,紫贻贝机体的基因表达量与代谢物含量差异,以解析PSTs胁迫下紫贻贝机体的炎症反应机制。结果表明,紫贻贝暴露于PSTs后表达的基因和代谢物均发生显著变化,其中差异表达基因17232个,差异代谢物341个。基于联合分析,差异表达基因与差异代谢物显著富集在花生四烯酸和谷胱甘肽代谢通路中,基因PLA2G2F、ALOX5、TBXAS1以及代谢物ARA、PGH₂、TXA₂、LTA₄、5-HETE对贻贝机体的促炎反应发挥重要作用;而基因GPX4、CYP2J2和代谢物15-HETE、GSH则调节机体炎症的消退。本研究揭示花生四烯酸相关通路在贻贝机体炎症反应过程中具有重要的调控作用,为下一步深度揭示PSTs胁迫下贻贝机体炎症网络化响应机制提供了研究基础。     

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.     

A multidisciplinary study of the Cape Peloro brackish area (Messina, Italy): characterisation of trophic conditions, microbial abundances and activities

In the framework of the VECTOR DIVCOST Project, a 2-year investigation was started in 2006, with the aim of testing the sensibility of microbial parameters to environmental changes and of assessing whether they can provide information about functional changes in the carbon cycle. The investigation was performed in the surface waters of two small brackish ponds (Ganzirri and Faro), located in the Cape Peloro transitional area (Sicily, Italy). The seasonal changes in both the microbial compartment [bacterioplankton, vibrios, exoenzymatic hydrolysis of proteins and polysaccharides, bacterial secondary production (HBP) and community respiration] and the trophic state of suspended matter [total suspended matter (TSM), particulate organic carbon (POC), particulate organic nitrogen (PON), C/N] were analysed in relation to the hydrological characteristics [temperature, salinity, oxygen, fluorescence, NH₄, NO₂, NO₃, PO₄]. Despite marked differences in the nutritional input and the diversification in both carbon budget and trophic level, the two ponds show similar trends in many of the investigated factors, hardly influenced by seasonal variations. Temporally coupled trends were observed for some parameters (enzyme activities, vibrios abundances, respiratory activity), whereas others (POC, PON, heterotrophic bacterial production, bacterioplankton) showed a seasonal shift between the two lakes. The different behaviour found for the some biotic parameters suggests that their response to environmental conditions may be modulated differently between the two lakes, which, despite their spatial proximity and reciprocal connection, do not always show contemporaneous functional processes.     

The effects of paraquat on microsomal oxygen reduction and antioxidant defenses in ribbed mussels (Geukensia demissa) and wedge clams (rangia cuneata)

The hepatopancreae of the ribbed mussel, Geukensia demissa, and the wedge clam, Rangia cuneata, were examined for their ability to catalyze the reduction/oxidation cycling of the bipyridyl herbicide paraquat. In vitro studies indicated a dose-dependent increase in the rate of superoxide anion (O₂⁻) generation in microsomal fractions supplemented with NADPH; the highest concentration of paraquat employed (4 mm) elicited an 81 % increase in cytochrome c reduction in G. demissa and a 135% increase in R. cuneata. In both .species, cytochrome c reduction was inhibited by the addition of exogenous superoxide dismutase (SOD). For in vivo studies, a single application of paraquat (0·5, 1·0 or 2·0 mm) was added to aerated salt-water aquaria containing G. demissa. Biochemical analyses of antioxidant enzymes, reduced glutathione (GSH) and lipid peroxidation were performed in hepatopancreatic tissue after exposures of 6, 12, 24 and 36 h. Results support the hypotheses that these bivalves can activate redox cycling compounds and demonstrate in vivo responses typical of oxidative stress as observed in other organisms.     

Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF.     

The influence of salinity on the heat-shock protein response of Potamocorbula amurensis (Bivalvia)

For biomarkers to be useful in assessing anthropogenic impacts in field studies involving aquatic organisms, they should not be affected by naturally occurring changes in environmental parameters such as salinity. This is especially important in estuarine environments and for relatively unspecific biomarkers like heat-shock proteins (hsps, stress proteins). In this study, the heat-shock protein response was measured in the euryhaline clam, Potamocorbula amurensis, after exposure to a range of salinities reflecting normal and extreme environmental conditions in Northern San Francisco Bay, California. The ability to raise cellular hsp70 levels in response to heat-shock was significantly impaired in P. amurensis collected from a low (0.5 ppt) salinity field site, and after 14 day exposure to low salinity in the laboratory.     

Embryonic exposure to environmentally relevant concentrations of PCB126 affect prey capture ability of Fundulus heteroclitus larvae

Early life stages from a marine fish species, Fundulus heteroclitus, were exposed to sublethal doses of 3,3',4,4',5 pentachlorobiphenyl (PCB126) to evaluate its effects on ecologically relevant responses: growth and behavior. A few hours after fertilisation, eggs were treated topically with PCB126 (2.5-50 pg egg?1). Four days post-hatching (dph), morphological changes (body length and malformations), spontaneous locomotor activity (active swimming speed, rate of travel, % inactivity), prey capture ability (Artemia franciscana nauplii) and whole body EROD activity were evaluated in larvae. Untreated larvae collected at 0.5, 1, 2, 3, 4 dph were also examined. PCB126 did not increase the mortality or malformation rates. Body length and spontaneous locomotor activity were altered only in larvae treated with the highest dose. Treatment with PCB126 caused a dose-responsive reduction in prey capture ability (rate of decline in the number of Artemia) and induction of EROD activity. The lowest observed effective dose for both of these responses was 5.0 pg PCB126 egg?1 or 5.0 TCDD-toxic equivalents pg g?1 egg, using a TCDD-toxic equivalent factor of 0.005 and an egg mass of 5 mg. Prey capture efficiency (number of Artemia captured per feeding strike) was reduced at ≥ 10.0 pg egg?1. In untreated developing larvae, prey capture ability and efficiency increased as post-hatching development progressed and EROD activity remained low. The pattern of behavioral responses observed in PCB126-exposed Fundulus larvae differed from that observed in less-developed larvae indicating that other mechanisms than retarded development were involved. Behavioral dysfunction was a more sensitive response to PCB126 than morphological alterations and it occurred at environmentally relevant concentrations.     

黄河口现行流路入海水沙特征对流域的响应

基于1976-2012年利津站实测水沙资料,选取1976-1986年、1987-1999年及2000-2012年作为特征时段,探讨了黄河口现行入海水沙条件变化的阶段性特征,重点研究了入海泥沙粒度特征对入海水沙变化的响应情况。结果表明:(1)与1976-1986年年均入海流量1 020 m~3/s相比,1987-1999年与2000-2012年入海流量大幅度减小,分别为473.7 m~3/s和504.7 m~3/s;(2)与1976-1986年年均入海含沙量24.4 kg/m~3相比,1987-1999年大幅度减小至15.9 kg/m~3,2000-2012年仅为4.1 kg/m~3;(3) 1976-1986年入海悬沙中值粒径为0.022 mm,1987-1999年减小至0.016 mm,2000-2012年又变粗至0.021 mm,反映水库正常运行一段时间后,下游河床冲刷造成入海泥沙粗化;(4) 2000年后利津站与花园口站悬沙粒度特征变化趋势趋于一致。研究还揭示了利津站床沙特征与流域前4年来沙系数存在较好相关性,利津床沙粒径呈现随来沙系数减少而增大的趋势,表明流域来沙量减少会造成利津站床沙粗化。