Cloning of the major 3-MC inducible phase I and phase II detoxification enzymes of plaice liver

cDNA's coding for cytochrome P4501A1 (CYP1A1), phenol UDP-glucuronosyltransferase (UDPGT) and glutathione S-transferase (GST-A) were cloned and sequenced from an expression library prepared from the liver of a 3-methylcholanthrene (3-MC) induced plaice. Plaice CYP1A1 and Phenol UDPGT display a high degree of structural conservation with homologous mammalian isoforms and their mRNAs were shown to be highly induced in liver after 3-MC treatment of fish. Expression of plaice GST-A, which displays closer homology to GSTs from plants and invertebrates than those of mammals, is repressed after 3-MC treatment.     

Pollutant-induced cell injury in fish liver: Use of fluorescent molecular probes in live hepatocytes

This study was conducted to test whether live cells from the liver of fish could be used to detect early changes that are indicative of pollutant-induced liver damage. Low-molecular-weight fluorescent molecular probes were inserted into isolated hepatocytes from dab (Limanda limanda) from a contaminated site and a reference site in the North Sea. These included bioprobes for endoplasmic reticulum (ER), cytochrome P-450-associated 7-ethoxyresorufin-O-deethylase (EROD), oxyradicals, reduced glutathione (GSH) and microtubules (MT). Endocytosis of Texas Red-albumin was used as an integrated indicator of hepatocyte performance. Findings showed increases in ER-associated fluorescence, EROD and oxyradical generation, with a marked decrease in endocytosis in hepatocytes from fish caught at the contaminated site. These results indicate that fish from the historically contaminated site were impacted by organic xenobiotics which induced the biotransformation system, radical production and cell injury.     

Tissue slice technology for assessing alterations in fish hepatic phase I and phase II XME activity

Alterations to hepatic xenobiotic metabolizing enzymes (XMEs) is an important biomarker of contaminant exposure in aquatic toxicology. Measurement of XMEs in fish liver slices in vitro is an emerging tool for examining enzyme activity and response within the intact 3-D architecture of the liver tissue. We examined integrated phase I/phase II, and phase II metabolism of XMEs from liver slices in control and B[a]P-treated rainbow trout and channel catfish. Fluorescent assay substrates to measure rates of metabolism included 7-methoxycoumarin (7-MC), 7-ethoxycoumarin (7-EC) and 7-hydroxycoumarin (7-HC). Time-dependent increases in metabolism, and a lower rate of 7-MC metabolism compared with 7-EC metabolism, were observed at all time points for both fish species. In rainbow trout, B[a]P pretreatment caused a 10-fold increase in phase I metabolism of both 7-MC and 7-EC, and a 1.6-fold increase in phase II metabolism of 7-HC. Phase I activity in channel catfish was not notably altered by B[a]P pretreatment. However, B[a]P pretreatment in channel catfish caused a 48% decrease in phase II metabolism of 7-HC. These results indicate differences in baseline and B[a]P-altered XME profiles between rainbow trout and channel catfish.     

In vitro alternatives to whole animal testing. Comparative cytotoxicity studies of divalent metals in established cell lines derived from tropical and temperate water fish species in a neutral red assay

A simple vital dye (neutral red) uptake method for investigation of the acute toxicity of seven divalent metal cations to cell lines derived from the turbot (TF) and bluegill (BF-2) was evaluated. The method was extremely reproducible between replicate experiments on a week to week basis as well as being reproducible between different laboratories. Whilst there were some species-dependent differences in the relative toxicities of the metals there were good correlations between toxicity and the chemical softness (Σp) of the metal ions and also between in vivo and in vitro toxicities. These studies demonstrate that this simple and relatively inexpensive technique carried out in 96-well microplates may be extremely useful for evaluation of structure-toxicity relationships between related groups of compounds.     

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis × M. crysops), and bluegill (Lepomis macrochuris) (n = 8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.     

The effect of long-term depuration on phase I and phase II biotransformation in mullets (Mugil cephalus) chronically exposed to pollutants in River Douro Estuary, Portugal

Pollutants such polycyclic aromatic hydrocarbons (PAHs) are released into the environment by urban communities and industries and the enzymes that catalyse the biotransformation of pollutants play a key role regarding the accumulation of these compounds in fish species inhabiting these areas. In this study the relationship between phase I (EROD activity) and phase II (GST activity) and PAH metabolites was measured in grey mullet (Mugil cephalus) after capture in the Douro estuary, and after long-term depuration in an unpolluted laboratory environment. The results showed a significant decrease in EROD activity after 1 month and in bile metabolites after 4 months in captivity, with both maintaining reduced levels at 4 and 8 months depuration. Liver GST activity did not showed significant changes. This study provides evidence that Douro estuary waters contain bioavailable PAHs that can be associated with the induction of cytochrome P450, and that mullets have the ability to metabolise and eliminate PAHs.     

Studies in seawater and lake water on interactions of trace metals with humic substances isolated from marine and estuarine sediments: II. Voltammetric investigations on trace metal complex formation in the dissolved phase

In seawater and lake water the complexation of several heavy metals, present in the dissolved state at relevant trace levels, with humic substances isolated from two marine and an estuarine sediment has been studied by differential pulse anodic stripping voltammetry (DPASV) at a hanging mercury drop electrode (HMDE). Taking Zn(II) as an example, the aim of this study was to gain direct information on the general level of importance of humic substances for the speciation of certain heavy metals in fresh and saline waters.In seawater humic acids originating from different sediments have very similar complexing properties for Zn(II), decreasing slightly in the order HAN > HAL > HAM. In lake water HAL is most efficient for complexing Zn(II), followed by HAN and HAM.In both types of natural waters, the sedimentary fulvic acid is less efficient in complexing Zn(II) than the respective humic acid from the same site. In general, the complexing efficiency decreases in the order EDTA > NTA > humic acids > fulvic acid.The problem of humic acid adsorption at the electrode, which somewhat limits the investigation of Pb(II) and Cd(II), and the impact of pH on the amount of complexed Zn(II) are also discussed.The findings provide further direct evidence for the conclusion previously drawn from existing complexation data that because of the rather low levels of dissolved humics in large parts of the oceans, the complexation efficiency of humics for Cd, Pb and Zn is too low to affect the speciation pattern of these three heavy metals.     

Use of inhibitors for coastal bacteria and phytoplankton: Application to nitrogen uptake measurement

For several decades, prokaryotic and eukaryotic inhibitors have been used to exclude bacteria from microalgal cultures and for investigating prey-predator relationships. Recently there has been considerable interest in using specific inhibitors for studying the interactions between bacteria and phytoplankton, by selective repression of either organism’s activity. The effectiveness of chemical inhibitors must be tested before applying them to natural communities to partition metabolic activities between functional groups. Six different antibiotics selected from the most commonly reported in the literature were tested, at concentrations varying from 12.5 to 100 mg L⁻¹, for their effect on bacterial growth and functional diversity of natural communities from Mediterranean coastal waters. Penicillin and streptomycin each at a final concentration of 100 mg L⁻¹ significantly reduced bacterial growth within 2 h. There was a greater impact on bacterial functional diversity when both antibiotics were mixed together. This mixture did not have any significant effect on the growth of selected cultured phytoplankton strains, whereas the eukaryote inhibitor cycloheximide at 100 mg L⁻¹ reduced growth within 2 h of incubation. The penicillin–streptomycin mixture and cycloheximide alone successfully partitioned NH₄⁺ and NO₃⁻ uptake between bacteria and phytoplankton bi-weekly sampled in a coastal lagoon in Autumn, where bacterial contribution to total NH₄⁺ and NO₃⁻ uptake averaged 46 and 41%, respectively. The use of specific inhibitors may be a valuable method for studying interactions, such as competition and mutualism, or lack of interaction between the different components of microbial communities and could be used to study their relative importance in biogeochemical fluxes.     

Use of suppressive subtractive hybridization and cDNA arrays to discover patterns of altered gene expression in the liver of dihydrotestosterone and 11-ketotestosterone exposed adult male largemouth bass (Micropterus salmoides)

In this study male largemouth bass (LMB) were exposed to the naturally occurring androgens, dihydrotestosterone (DHT) or 11-ketotestosterone (11-KT) in order to identify genes that are differentially regulated by these steroid hormones. Using subtractive hybridization on livers of fish treated with DHT against vehicle control, many novel LMB genes were cloned. These genes were added to our gene library and arrayed. Six genes were up-regulated and five were down-regulated by both androgens. But, each androgen also regulated specific genes. One gene that was identified as a potential androgen marker was spermidine-spermine-N(1)-acetyltransferase that was up-regulated by both androgens. Determining which genes are responsive to natural androgens will help to identify biochemical pathways that are impacted.     

The AquaDEB project (phase I): Analysing the physiological flexibility of aquatic species and connecting physiological diversity to ecological and evolutionary processes by using Dynamic Energy Budgets

The European Research Project AquaDEB (2007–2011, http://www.ifremer.fr/aquadeb/) is joining skills and expertise of some French and Dutch research institutes and universities to analyse the physiological flexibility of aquatic organisms and to link it to ecological and evolutionary processes within a common theoretical framework for quantitative bioenergetics [Kooijman, S.A.L.M., 2000. Dynamic energy and mass budgets in biological systems. Cambridge University Press, Cambridge]. The main scientific objectives in AquaDEB are i) to study and compare the sensitivity of aquatic species (mainly molluscs and fish) to environmental variability of natural or human origin, and ii) to evaluate the related consequences at different biological levels (individual, population, ecosystem) and temporal scales (life cycle, population dynamics, evolution). At mid-term life, the AquaDEB collaboration has already yielded interesting results by quantifying bio-energetic processes of various aquatic species (e.g. molluscs, fish, crustaceans, algae) with a single mathematical framework. It has also allowed to federate scientists with different backgrounds, e.g. mathematics, microbiology, ecology, chemistry, and working in different fields, e.g. aquaculture, fisheries, ecology, agronomy, ecotoxicology, climate change. For the two coming years, the focus of the AquaDEB collaboration will be in priority: (i) to compare energetic and physiological strategies among species through the DEB parameter values and to identify the factors responsible for any differences in bioenergetics and physiology; and to compare dynamic (DEB) versus static (SEB) energy models to study the physiological performance of aquatic species; (ii) to consider different scenarios of environmental disruption (excess of nutrients, diffuse or massive pollution, exploitation by man, climate change) to forecast effects on growth, reproduction and survival of key species; (iii) to scale up the models for a few species from the individual level up to the level of evolutionary processes.     

4-tert-octylphenol and 17beta-estradiol applied by feeding to flounder Platichthys flesus: induction of vitellogenin and accumulation in tissues

In order to simulate a possible natural administration route of xenoestrogens male flounder Platichthys flesus were exposed via the diet to the alkylphenol 4-tert-octylphenol. Treatment with 4-tert-octylphenol (10, 50 and 100 mg OP kg(-1) BW) or 17beta-estradiol (0.05 mg kg(-1) BW) every second day during a period of 11 days resulted in a significant increase in plasma vitellogenin (Vtg) concentrations. The induction of Vtg was greatest in the fish receiving 50 mg OP kg-' BW. A significant accumulation of 4-tert-octylphenol was found in liver and muscle tissue of the OP treated groups. The tissue concentrations of 4-tert-octylphenol and the plasma vitellogenin concentration were positively correlated. The results show that 4-tert-octylphenol accumulates in liver and muscles of flounder P. flesus, and exerts estrogenic effects such as vitellogenin induction.     

Studies in seawater and lake water on interactions of trace metals with humic substances isolated from marine and estuarine sediments: I. Characterisation of humic substances

Two humic acids, HAL and HAN, isolated from marine sediments (Adriatic and Norwegian Sea) and humic (HAM) and fulvic (FAM) acids isolated from an estuarine sediment (Mahakam, Borneo) were characterized.The following characteristics: carbon content, hydrophobicity, degree of condensation and the content of aliphatic groups, are most pronounced for HAN, less pronounced for HAL and the least pronounced for HAM. The abundance of trace metals in the humic acids decreases in the following order: Al > Cu > Zn > Pb > Cd.Fulvic acid (FAM) has a lower carbon and a higher oxygen content than humic acids. It is hydrophilic and hardly any aliphatic groups were recorded. At pH 7 the predominant fraction of FAM has the apparent molecular weight of 20 000 daltons while the humic acids are of higher molecular weight.     

Use of suppressive subtractive hybridization and cDNA arrays to discover patterns of altered gene expression in the liver of dihydrotestosterone and 11-ketotestosterone exposed adult male largemouth bass (Micropterus salmoides)

In this study male largemouth bass (LMB) were exposed to the naturally occurring androgens, dihydrotestosterone (DHT) or 11-ketotestosterone (11-KT) in order to identify genes that are differentially regulated by these steroid hormones. Using subtractive hybridization on livers of fish treated with DHT against vehicle control, many novel LMB genes were cloned. These genes were added to our gene library and arrayed. Six genes were up-regulated and five were down-regulated by both androgens. But, each androgen also regulated specific genes. One gene that was identified as a potential androgen marker was spermidine-spermine-N¹-acetyltransferase that was up-regulated by both androgens. Determining which genes are responsive to natural androgens will help to identify biochemical pathways that are impacted.     

大气环流优势模态对北极海冰变化的响应Ⅰ.北极涛动

利用美国冰雪中心海冰密集度数据,分析了1979-2012年北极海冰面积的时间变化特征,发现北极海冰具有显著的年代际变化特征,分别在1997和2007年前后存在两次年代际转型突变点,相应的大气环流优势模态——北极涛动(AO)也存在显著的时空变化。1979-1996年阶段海冰下降趋势较弱并以较强的年际振荡为主,AO模态较强且显示出低频振荡特征;1997-2006年阶段北极海冰快速减退趋势占优,同时伴随着较弱的年际振荡,AO模态减弱且振荡周期缩短;2007-2012年阶段海冰范围较快下降同时具有极强的年际振荡,方差变化是前两个阶段的2~3倍,AO不仅强度加强,空间结构也发生了变化,极涡中心分别向格陵兰岛和白令海峡一侧延伸,这种结构有利于极地冷空气入侵欧洲和北美。利用ECHAM5大气模式进行的数值试验结果也证实了较强振荡的海冰强迫对AO模态的改变具有决定作用。     

Late glacial to Holocene sea-level changes in the Sea of Marmara: new evidence from high-resolution seismics and core studies

The late glacial to Holocene sedimentary record of the northern shelf of the Sea of Marmara (SoM) has been documented by detailed seismo-, chrono-, and biostratigraphic analyses using sub-bottom (Chirp) profiles and sediment cores. During MIS 3 and the main part of MIS 2 (60–15 ¹⁴C ka b.p.), disconnection from the Mediterranean and Black seas together with a dry climate resulted in a regression in the SoM, when the Sea was transformed into a brackish lake. The river incisions below 105 m water depth along the northern shelf took place during the last glacial maximum, when the lake level was modulated by stillstands at −98 and −93 m. The post-glacial freshwater transgressive stage of the Marmara ‘Lake’ occurred between 15 and 13.5 ¹⁴C ka b.p., leading to a rise in water level to −85 m by 13.0 ¹⁴C ka b.p., as evidenced by broad wave-cut terraces along the northern shelf. Since 12 ¹⁴C ka b.p., high-frequency sea-level fluctuations have been identified at the SoM entrance to the Strait of İstanbul (SoI). Thus, wave-cut terraces have been recorded at water depths of −76 and −71 m that, according to an age model for core MD04-2750, have ages of 11.5 and 10.5 ¹⁴C ka b.p., respectively. Ancient shoreline at −65 m along the northern shelf presumably formed soon after the Younger Dryas (YD) at ca. 10.1 ¹⁴C ka b.p. Moreover, there is compelling evidence of Holocene outflow from the Sea of Marmara to the Black Sea. At the SoM entrance to the SoI, the existence of bioherms on the reflector surface together with abundant Brizalina spathulata and Protoglobulimina pupoides in a core suggests a return to higher salinities due to strong Mediterranean water incursion into the SoM at ∼8.8 ¹⁴C ka b.p. This finding is consistent with earlier suggestions that, after the YD, the Black Sea was flooded by outflow from the SoM as a result of global sea-level rise.     

Biogeochemical responses to late-winter storms in the Sargasso Sea,II: Increased rates of biogenic silica production and export

Previous studies measuring biogenic silica production in the Sargasso Sea, all conducted when no phytoplankton bloom was in progress, have reported a mean rate of 0.4 mmol Si m^?2 d^?1 and maximum rate of 0.9 mmol Si m^?2 d^?1, the lowest rates yet recorded in any ocean habitat. During February/March of 2004 and 2005 we studied the effects of late-winter storms prior to seasonal stratification on the production rate, standing stock and vertical export of biogenic silica in the Sargasso Sea. In 2004, alternating storm and stratification events provided pulsed input of nutrients to the euphotic zone. In contrast, nearly constant storm conditions in 2005 caused the mixed layer to deepen to ～350 m toward the end of the cruise. Biogenic silica production rates in the upper 140 m were statistically indistinguishable between years, averaging ～1.0 mmol Si m^?2 d^?1. In early March 2004, a storm event entrained nutrients into the euphotic zone and, upon stabilization, vertically integrated biogenic silica in the upper 140 m nearly doubled in 2 days. Within 4 days, 75–100% of the accumulated biogenic silica was exported, sustaining a flux to 200 m of ～0.5 mmol Si m^?2 d^?1 (4× greater than export measured during February and March in the mid-1990s). In 2005, destabilization without stratification increased biogenic silica flux at 200 m up to two-fold above previously measured export in late winter, with little or no increase in water-column biogenic silica. Despite comprising <5% of total chlorophyll, diatoms accounted for an estimated 25–50% of the nitrate uptake in the upper 140 m and 35–97% of the particulate organic nitrogen export from the upper 200 m during both cruise periods. These previously unobserved brief episodes of diatom production and export in response to late-winter storms increase the estimated production and export of diatom-derived material in the Sargasso Sea in late winter by >150%, and increase estimated annual biogenic silica production in this region by ～8%.     

Application of a sensitive chemiluminescent technique for comparison of cytochrome P4501A induction in hepatic and intestinal tissues of fish exposed to bleached kraft mill effluent

In conjunction with an environmental assessment of biologically-treated bleached kraft mill effluent (BKME) in a Western Canadian river, data indicated that lipophilic compounds were transferred to the mountain whitefish Prosopium williamsoni through ingestion of filter-feeding benthic caddisflies. P4501A induction was correlated with lipophilic body burdens, not with indices of recent BKME exposure. P4501A contents in hepatic and intestinal tissues of BKME-exposed whitefish were compared during a follow-up collection. Ethoxyresorufin O-deethylase (EROD) activity was not detectable in intestines; P-450 spectral analysis indicated denaturation had occurred. Use of enhanced chemiluminescence (ECL) immunoblotting achieved at least 10-fold greater sensitivity over colorimetric methods and indicated that some intestines did contain P4501A protein. No correlation of intestinal P4501A with hepatic EROD activity or P4501A content was found. Application of the ECL technique significantly improves the analytical detection limits of P4501A immunoblotting. Analysis of historical samples from this site will further probe water-borne versus dietary routes of uptake of BKME-related P4501A inducers in this species.