Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

欧洲鳗鲡胃肠道激素免疫组织化学的研究

用免疫组织化学技术研究了9种哺乳动物胃肠道激素抗体在正常生长欧鳗和僵鳗胃肠道的定位分布,研究结果表明,5－HT,SOM,GAS,α－1－AT和P物质免疫活性内分泌细胞存在于正常鳗和僵鳗胃肠道中,其余4种抗体显阴性反应,文中讨论了胃肠道激素在僵鳗成因中可能的生理作用。     

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.     

Endogenously-mediated, pretranslational suppression of cytochrome P4501A in PCB-contaminated flounder

Gonadally mature fish display strong sex-related differences in the content and activity of P4501A, the major polynuclear aromatic hydrocarbon-inducible P450 form in teleosts. Such differences appear related to plasma levels of the female sex steroid, estradiol (E₂); however, neither the mechanism of estradiol suppression of P4501A nor the capacity for hormonal regulation to overcome P4501A induction by high concentrations of potent inducers are known. Gonadally mature flounder (Pseudopleuronectes americanus) were collected from Fox Island (FI), Rhode Island, a reference site, and New Bedford Harbor (NB), Massachusetts, a site highly contaminated with polychlorinated biphenyls (PCBs). Differences in flounder P4501A expression were determined at the level P4501A catalytic activity (measured as ethoxyresorufin-O-deethylase, EROD), P4501A protein content (immunoquantitated), and P4501A mRNA content (by Northern blot) as they relate to sex, reproductive status, and hepatic PCB content. Our results confirm that suppression of P4501A in gonadally mature female fish is probably due, at least in part, to elevated E₂ titers, and demonstrate that such suppression occurs at a pretranslational level and, further, that endogenous regulation of P4501A expression can ‘override’ exogenous regulation by even high concentrations of P4501A inducers.     

The Ah receptor in marine animals: phylogenetic distribution and relationship to cytochrome P4501A inducibility

In mammals, the induction of cytochrome P4501A forms by chlorinated dibenzo-p-dioxins, chlorinated dibenzofurans, and halogenated biphenyls is under control of a soluble protein known as the Ah (aromatic hydrocarbon) receptor. Little is known about the presence and properties of the Ah receptor in other vertebrate and invertebrate species. In these studies, we sought evidence for an Ah receptor in the liver or liver-equivalent of 20 species of marine and freshwater animals, using the photoaffinity ligand 2-azido-3-[¹²⁵I] iodo-7,8-dibromodibenzo-p-dioxin (N₃[¹²⁵I]Br₂DD). Specific labeling of cytosolic proteins by N₃[¹²⁵I]Br₂DD was observed in seven species of teleost and elasmobranch fish, in PLHC-I fish hepatoma cells, and in beluga whales. No specifically labeled proteins were found in cytosol from two species of agnathan fish nor in any of nine invertebrate species representing eight classes of four phyla. The presence or absence of specifically labeled polypeptides corresponds with the inducibility of cytochrome P4501A and sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related planar halogenated aromatic hydrocarbons in many of these groups. Thus, Ah receptor function may have arisen early invertebrate evolution and has been conserved from elasmobranch and teleost fish to mammals.     

Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in nearshore demersal fishes

Three biomarkers of hydrocarbon exposure, CYP1A in liver vascular endothelium, liver ethoxyresorufin O-deethylase (EROD), and biliary fluorescent aromatic compounds (FACs), were examined in the nearshore fishes, masked greenling (Hexagrammos octogrammus) and crescent gunnel (Pholis laeta), collected in Prince William Sound, Alaska, 7-10 years after the Exxon Valdez oil spill (EVOS). All biomarkers were elevated in fish collected from sites originally oiled, in comparison to fish from unoiled sites. In 1998, endothelial CYP1A in masked greenling from sites that were heavily oiled in 1989 was significantly higher than in fish collected outside the spill trajectory. In 1999, fishes collected from sites adjacent to intertidal mussel beds containing lingering Exxon Valdez oil had elevated endothelial CYP1A and EROD, and high concentrations of biliary FACs. Fishes from sites near unoiled mussel beds, but within the original spill trajectory, also showed evidence of hydrocarbon exposure, although there were no correlations between sediment petroleum hydrocarbon and any of the biomarkers. Our data show that 10 years after the spill, nearshore fishes within the original spill zone were still exposed to residual EVOS hydrocarbons.     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals

Information about the expression of CYP1A in wildlife species is essential for understanding the impact of organochlorine exposure on the health status of an exposed population. Therefore, we aimed at characterising a putative CYP1A enzyme expression in both hepatic and extrahepatic tissues of ringed and grey seals from the Baltic Sea and from less polluted waters. The cellular localisation of CYP1A was identified using a monoclonal antibody against scup P4501A1 (MAb 1-12-3). Immunohistochemical staining showed the highest level of CYP1A expression in liver hepatocytes, and the second highest level in the endothelial cells of capillaries and larger blood vessels in the liver and other organs. The most frequent and strongest staining was found in Baltic ringed seals. Although CYP1A-positive staining was observed in only a few tissues in the other seal populations, it was more intense in Baltic grey seals than in Canadian grey seals. The CYP1A enzyme activity, expressed as ethoxyresorufin O-deethylation (EROD), followed a similar tissue distribution and geographical pattern as the immunohistochemistry with clearly elevated EROD activities in most tissues of both Baltic seal populations. Immunochemical characterisation by immunoblotting confirmed the presence and elevation pattern of a putative CYP1A protein in ringed and grey seals, supporting our findings using other methods. The evenly distributed elevation of CYP1A expression among most of the tissues examined indicates that Baltic seals are exposed to CYP1A inducing agents affecting the whole body. This may result in an increased or decreased toxic potential of foreign substances, which may ultimately determine the biological effects of the contaminants.     

Variation in levels of cytochrome P4501A, 2B, 2E, 3A and 4A-immunopositive proteins in digestive gland of indigenous and transplanted mussel Mytilus galloprovincialis in Venice Lagoon, Italy

Mytilus galloprovincialis digestive gland microsomes were prepared from (i) indigenous populations sampled from a clean reference (Lido) and an urban-contaminated site (Salute) and (ii) mussels transplanted for up to 3 weeks from Lido to an industrial-contaminated site (CVE) in Venice Lagoon, Italy. Western blot analysis was performed using antibodies to five mammalian or fish CYP forms (1A, 2B, 2E, 3A, 4A). Simultaneously run M. edulis digestive gland partially purified CYP aided identification of immunopositive bands. Levels of CYP1A, CYP2E, and CYP4A-immunopositive proteins were 50 to 300% higher in indigenous M. galloprovincialis from Salute compared to Lido (p < 0.05). Three weeks after transplantation to CVE, levels of only the CYP1A-immunopositive protein were determined to be higher (63%) than levels for Lido (p < 0.05), indicating that anti-CYP1A shows greater specificity for a contaminant-inducible CYP form than the other antibodies.     

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.     

Seasonal and interannual variability of carbon cycle in South China Sea: A three-dimensional physical-biogeochemical modeling study

The South China Sea (SCS) exhibits strong variations on seasonal to interannual time scale, and the changing Southeast Asian Monsoon has direct impacts on the nutrients and phytoplankton dynamics, as well as the carbon cycle. A Pacific basin-wide physical-biogeochemical model has been developed and used to investigate the physical variations, ecosystem responses, and carbon cycle consequences. The Pacific basin-wide circulation model, based on the Regional Ocean Model Systems (ROMS) with a 50-km spatial resolution, is driven with daily air-sea fluxes derived from the National Centers for Environmental Prediction (NCEP) reanalysis between 1990 and 2004. The biogeochemical processes are simulated with the Carbon, Si(OH)₄, Nitrogen Ecosystem (CoSINE) model consisting of multiple nutrients and plankton functional groups and detailed carbon cycle dynamics. The ROMS-CoSINE model is capable of reproducing many observed features and their variability over the same period at the SouthEast Asian Time-series Study (SEATS) station in the SCS. The integrated air-sea CO₂ flux over the entire SCS reveals a strong seasonal cycle, serving as a source of CO₂ to the atmosphere in spring, summer and autumn, but acting as a sink of CO₂ for the atmosphere in winter. The annual mean sea-to-air CO₂ flux averaged over the entire SCS is +0.33 moles CO₂ m⁻²year⁻¹, which indicates that the SCS is a weak source of CO₂ to the atmosphere. Temperature has a stronger influence on the seasonal variation of pCO₂ than biological activity, and is thus the dominant factor controlling the oceanic pCO₂ in the SCS. The water temperature, seasonal upwelling and Kuroshio intrusion determine the pCO₂ differences at coast of Vietnam and the northwestern region of the Luzon Island. The inverse relationship between the interannual variability of Chl-a in summer near the coast of Vietnam and NINO₃ SST (Sea Surface Temperature) index in January implies that the carbon cycle and primary productivity in the SCS is teleconnected to the Pacific-East Asian large-scale climatic variability.     

Seasonal and annual variability in particle fluxes in the Gulf of California: A response to climate forcing

A six-year time series of sediment trap samples is used to document seasonal and interannual changes in particle fluxes in Guaymas Basin, Gulf of California. Sedimentation in this region of the Gulf of California is dominated by two seasonally varying components: biogenic silica during the late fall–spring and lithogenic material during the summer. This variability in sediment fluxes is a direct response to the seasonally reversing monsoon climate in this region. In addition, this seasonal variability in particle flux to the seafloor combined with the lack of bioturbation results in the formation of sediment laminae or varves in Guaymas Basin. Observed interannual differences in the magnitude of sediment fluxes appear to be related to ENSO. These findings indicate that biogenic sediment fluxes, and hence primary productivity, are higher during non-ENSO years in the central Gulf of California. A decrease in the carbonate: opal flux ratio from 1991 to 1996 indicates that diatoms became an increasingly important component of the plankton during this period and further supports our findings that productivity in Guaymas Basin is higher during non-ENSO years.     

Quantification of cytochrome P4501A1 and catalytic activities in liver microsomes of isosafrol- and β-Naphthoflavone-treated rainbow trout (Oncorhynchus mykiss)

The effects of isosafrol (ISF) or β-naphthoflavone (βNF) treatments on cytochrome P450 (P450) levels in rainbow trout liver were investigated using immunochemical and catalytic techniques. The discrepancies in catalytic activities and ELISA quantification of rainbow trout P4501A1 protein levels between ISF- and βNF-treated fish indicate that important differences exist between the responses induced by βNF and ISF treatments in the rainbow trout liver.     

Scales of predator detection behavior and escape in Fissurella limbata: A field and laboratory assessment

The consumptive effects of predators are widely acknowledged, but predation can also impact prey populations through non‐consumptive effects (NCEs) such as costly antipredator behavioral responses. The magnitude of antipredator behavioral responses by prey is determined by an assessment of risk using sensory cues, which in turn is modulated by the environmental context. We studied the detection behavior and escape response of the keyhole limpet Fissurella limbata from the predatory sea star Heliaster helianthus. Through laboratory and field experimental trials, we quantified the distance and time of predator detection behavior by the prey, and measured their active escape responses when elicited. We found that predator detection by the limpet was chiefly mediated by distance, with experimental individuals capable of detecting predator presence effectively up to distances of at least 50 cm in the field and 70 cm under laboratory conditions. Our results indicate that this prey species is able to evaluate the proximity of its predator and use it as an indication of predation risk; therefore, predator–prey distance appears to be a primary predictor of the magnitude of the antipredator response. Given the tight relationship between predator distance and prey movement and the important role herbivores can play, particularly in this ecosystem, we expect that NCEs will cascade to the patterns of abundance and composition of rocky shore communities through changes in prey foraging behavior under risk.     

Seasonal and interannual variability of oceanic carbon cycling in the western and central tropical-subtropical pacific: A physical-biogeochemical modeling study

The oceanic carbon cycle in the tropical-subtropical Pacific is strongly affected by various physical processes with different temporal and spatial scales, yet the mechanisms that regulate air-sea CO₂ flux are not fully understood due to the paucity of both measurement and modeling. Using a 3-D physical-biogeochemical model, we simulate the partial pressure of CO₂ in surface water (pCO_2sea) and air-sea CO₂ flux in the tropical and subtropical regions from 1990 to 2004. The model reproduces well the observed spatial differences in physical and biogeochemical processes, such as: (1) relatively higher sea surface temperature (SST), and lower dissolved inorganic carbon (DIC) and pCO_2sea in the western than in the central tropical-subtropical Pacific, and (2) predominantly seasonal and interannual variations in the subtropical and tropical Pacific, respectively. Our model results suggest a non-negligible contribution of the wind variability to that of the air-sea CO₂ flux in the central tropical Pacific, but the modeled contribution of 7% is much less than that from a previous modeling study (30%; McKinley et al., 2004). While DIC increases in the entire region SST increases in the subtropical and western tropical Pacific but decreases in the central tropical Pacific from 1990 to 2004. As a result, the interannual pCO_2sea variability is different in different regions. The pCO_2sea temporal variation is found to be primarily controlled by SST and DIC, although the role of salinity and total alkalinity, both of which also control pCO_2sea, need to be elucidated by long-term observations and eddy-permitting models for better estimation of the interannual variability of air-sea CO₂ flux.