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.     

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.     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

EROD activities of liver in Mugil so-iuy exposed to benzo (a)pyrene, pyrene and their mixture

Abstract-The effects on hepatic EROD (7-ethoxyresorufin O-deethylase) in Mugil so-iuy exposedto benzo(a)pyrene (BaP), pyrene and their mixtures of equal concentration were investigated, at con-centrations of 0.1, 1.0, 5.0, 10.0, 50.0 μg/dm~3, in experimental condition. Time-effects and dose-response of the biochemical indexs were observed. The results showed that the hepatic EROD activitieswere induced by the exposure of BaP, pyrene and their mixtures at high concentration. Dose-responseconnections were that the hepatic EROD activities were elevated with increasing concentration of the pol-lutants. The combined effect of BaP and pyrene at 1:1 concentration ratio on hepatic EROD activity wasantagonism.     

Ethoxyresorufin-O-deethylase activity and fixed wavelength fluorescence detection of PAHs metabolites in bile in turbot (Scophthalmus maximus L.) Exposed to a dispersed topped crude oil in a Continuous Flow System

Long term effects of sublethal concentrations of oil on the marine environment have become of general concern. Cytochrome P4501A activity (EROD) in liver and fixed wavelength fluorescence detection of PAHs metabolites (FF) have in this study been used as biomarkers for dispersed oil exposure on a long term period of juvenile turbot (Scophthalmus maximus L.). A Continuous Flow System was used to carry out the study. The fish were continuously exposed to 0.125, 0.5 or 2.0 mg litre⁻¹ dispersed topped crude oil for 6, 15, 24 h, 4 and 21 days followed by a 9 days recovery period in clean seawater. No induction of the cytochrome P4501A was measured. A maximum level in bile metabolites (4- to 5-fold) was recorded after 24 h of exposure revealing thereby a detoxification process, but a decline occurred from day 4 to day 21. This study demonstrated that FF detection of PAHs metabolites in bile could be a more sensitive biomarker than EROD activity in a long term exposure to sublethal concentration of oil.     

Effects of β-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 β-naphthoflavone (β-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 β-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 β-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 β-NF.     

原油WSF对三种海洋鱼类肝微粒体EROD活性的诱导和恢复的比较

研究了实验室条件下原油水溶性组分(WSF)暴露对黑鲷、黄鳍鲷和褐菖鲉肝微粒体EROD活性的剂量-效应,时间-效应和恢复过程。实验结果表明,在剂量诱导实验中,褐菖鲉肝EROD活性在原油WSF浓度为50μg/dm3时呈现生物统计学上的显著差异,而黑鲷和黄鳍鲷肝EROD活性在75μg/dm3时才呈现生物统计学上的显著差异;褐菖鲉肝EROD活性诱导倍数最高,但黑鲷的诱导浓度范围较广。在时间诱导实验中,在40μg/dm3原油WSF暴露下黄鳍鲷肝EROD活性在2 d时首先呈现显著差异;三种鱼肝EROD活性均在第4天达到最高,并呈现显著性变化,此后随着暴露时间的延长而逐渐下降并接近对照组水平。在恢复实验中三种鱼肝EROD活性下降并恢复到对照组水平。研究结果表明:对于石油污染物,黑鲷、黄鳍鲷和褐菖鲉肝EROD活性都可以作为污染生化效应监测指标,然而就三种鱼类比较而言,褐菖鲉最敏感,更适合于作为石油类污染及其生化效应,尤其是低剂量效应的监测生物。     

Lysosomal injury and MFO activity in the liver of flounder (Platichthys flesus L.) in relation to histopathology of hepatic degeneration and carcinogenesis

Flounder (Platichthys flesus L.), 18–48 cm total length were sampled in the mouth of the Elbe River during a 3-year interdisciplinary project ‘Fish Diseases in the Wadden Sea’. Lysosomal changes (lysosomal membrane stability) and the activity of biotransforming enzymes (MFOs/EROD) were measured parallel to investigations of ultrastructural changes in liver cells, tissue pathologies and macroscopically visible changes. The aim of the study was to investigate if these cyto- and biochemical parameters were able to reflect contaminant induced biological effects. Interlinking of the results of the MFO activity to the pathological alterations observed at the electron- and light-microscopic levels as well as during macroscopic inspection of identical individuals evidenced that the activity of the biotransformation enzymes (EROD) was not induced in healthy livers, increased considerably with the onset of liver changes and dropped again in those livers with degenerative, preneoplastic and neoplastic lesions. In contrast, concurrent studies of lysosomal membrane stability measured in parallel showed a decrease with the onset and progression of liver lesions from reversible to irreversible, neoplastic change.     

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.     

Distribution, condition, and growth of newly settled southern flounder (Paralichthys lethostigma) in the Galveston Bay Estuary, TX

Several flatfish species, including southern flounder (Paralichthys lethostigma) recruit to estuaries during early life. Therefore, evaluation of estuarine sites and habitats that serve as nurseries is critical to conservation and management. The present study used density data in conjunction with biochemical condition and growth measurements to evaluate settlement sites used by southern flounder in the Galveston Bay Estuary (GBE). In 2005, beam-trawl collections were made in three major sections of the GBE (East Bay, Galveston Bay, West Bay). Three sites were sampled in each bay. Within each sampling site, replicate collections were taken from three habitats: 1) marsh edge (< 1 m depth), 2) intermediate zone (10–20 m from marsh interface;  1 m depth), and 3) bay zone (typically > 100 m from marsh interface; depth > 1 m). Average size of southern flounder collected was 12–19 mm standard length, and peak densities occurred in January and February. Catch data indicated that densities of southern flounder were significantly greater in East Bay (2.75 per 100 m²) than in Galveston Bay (0.91 per 100 m²) or in West Bay (0.45 per 100 m²). Densities were statistically similar among habitats. Otolith-based estimates of age indicated that the majority of southern flounder collected were 35–45 days old and derived from early December to early January hatch-dates. Growth rates were similar among bays and among habitats, with the average growth rate being 0.40 mm day^− 1 (range: 0.21–0.76 mm day^− 1). RNA:DNA was above the established baseline value for nutritional stress, indicating that newly settled southern flounder in the GBE were in relatively high condition. Habitat-specific differences in RNA:DNA ratios were not observed; however, ratios were significantly lower in West Bay (average 8.0) than in East Bay (average 9.5) or in Galveston Bay (average 9.8), suggesting the condition of new recruits may vary spatially within the GBE. Findings from the current study suggest density and condition of newly settled southern flounder vary at the bay scale, suggesting that parts of GBE do not function equally as nurseries.     

Winter distribution of euphausiids (Euphausiacea) in the Barents Sea (2000–2005)

The purpose of the study is to analyze the state of the Barents Sea euphausiids populations in the warm period (2000–2005) based on the study of their structure dynamics and distribution under the influence of abiotic and biotic factors. For estimation of their aggregations in the bottom layer, the traditional method was used with the help of the modified egg net (0.2 m² opening area, 564 μm mesh size). The net is used for collecting euphausiids in the autumn–winter period when their activity is reduced, which results in high-catch efficiency. The findings confirmed the major formation patterns of the euphausiids species composition associated with climate change in the Arctic basin. As before, in the warm years, one can see a clear-cut differentiation of space distribution of the dominant euphausiids Thysanoessa genus with localization of the more thermophilic Thysanoessa inermis in the north-west Barents Sea and Thysanoessa raschii in the east. The major euphausiids aggregations are formed of these species. In 2004, the first data of euphausiids distribution in the northern Barents Sea (77–79°N) were obtained, and demonstrated extremely high concentrations of T. inermis in this area, with the biomass as high as 1.7–2.4 g m⁻² in terms of dry weight. These data have improved our knowledge of the distribution and euphausiids abundance during periods of elevated sea-water temperatures in the Barents Sea. The oceanic Atlantic species were found to increase in abundance due to elevated advection to the Barents Sea during the study period. Thus, after nearly a 30-year-long absence of the moderate subtropical Nematoscelis megalops in the Barents Sea, they were found again in 2003–2005. However in comparison with 1960, the north-east border of its distribution considerably shifted to 73°50′N 50°22′E. The portion of Meganyctiphanes norvegica also varied considerably—from 10% to 20% of the total euphausiids population in the warm 1950s–1960s almost to complete disappearing in 1970–1990s. The peak of this species’ occurrence (18–26%) took place in the beginning of warm period (1999–2000) after a succession of cold years. The subsequent reduction of the relative abundance of M. norvegica to 7% might have been mostly caused by fish predation during a period of low population densities of capelin. This high predation pressure may therefore have been mediated both by other pelagic fishes (i.e. herring, blue whiting, polar cod) but also by demersal fishes such as cod and haddock. Similar sharp fluctuations in the capelin stock (the major consumer of euphausiids) created marked perturbations in the food web in the Barents Sea in the middle 1980s and the early 1990s.     

Cytochrome P4501A1 induction in Kenai River sculpin (Cottus spp.) as a monitor of freshwater pollution effects

Freshwater sculpin were taken from various locations in the Kenai River to determine their in-vivo response to pollution exposure. The specific activity of cytochrome P4501A1 was determined using fluorescence detection of the 7-ethoxyresorufin-O-dealkylase (EROD) reaction. The EROD specific activity was found to be independent of river miles but appeared to relate to specific sites of urban runoff. Glutathione-S-transferase (GST) activities were also measured but showed no significant difference between sites. Cytochrome P450 laboratory induction studies were carried out on individuals collected from a pristine site. Five groups of 10 fish were dosed from 10–150 mg β-Naphthoftavone (βNF)/kg body weight. The response was linear from 10–50 mg βNF/kg body weight. Sculpin appear to have excellent potential as a sensitive indicator of xenobiotic exposure in a freshwater benthic habitat.     

Regulation of hepatic glutathione S-transferase expression in flounder

The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254^TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species.     

Seasonal Variations in Abundance and Size Composition of the Lobate Ctenophore Bolinopsis Mikado (Moser) in Tokyo Bay, Central Japan

Surveys of the abundance and size composition of the ctenophore Bolinopsis mikado were conducted in Tokyo Bay over a 5-year period from 1990 to 1994. B. mikado appeared throughout the year, and its mass occurrence was observed between August and November; annual maximum abundance ranged from 19 to 91 ind. m^–2. Water temperature seems to influence the seasonal variation of B. mikado abundance. Environmental conditions (e.g. rough waters due to a typhoon) and predation by the beroid ctenophore Beroe cucumis appear to affect annual variations of B. mikado abundance. Size frequency distributions of B. mikado indicated that its reproduction was most active in summer and fall but occurred throughout the year in Tokyo Bay. A sharp decline of the copepod population in August 1990 was probably due to predation by B. mikado which was very abundant at that time; its predatory impact was estimated to be 24 % day^–1.     

Interaction of CYP1A1 inducers in liver of rainbow trout and eelpout

The hepatic CYP1A1 (ethoxyresorufin-O-deethylase (EROD) and protein level) in rainbow trout and eelpout was induced by isosafrole, β-naphthoflavone, 3,3′,4,4′-tetrachlorobiphenyl and mixtures of two of the compounds. A potentiation effect of the CYP1A1 response was observed when isosafrole was given together with β-naphthoflavone, but not when isosafrole was given with 3,3′,4,4′-tetrachlorobiphenyl.     

Availability of cadmium and zinc from sewage sludge to the flounder, Platichthys flesus, via a marine food chain

This paper examines the potential availability to a demersal fish of cadmium and zinc associated with digested sewage sludge, via a food chain as well as directly from the sludge, and the tissue distribution and possible excretion of any accumulated cadmium and zinc. Radioactive tracer techniques were used in order to follow the food chain transfer of the metals. Flounder (Platichthys flesus) accumulated cadmium both directly from sludge (delivered in newly ingested unassimilated gutfuls in the amphipods) and in assimilated form from the tissues of the amphipod crustacean Corophium volutator that had been feeding on a sludge–sediment mixture. Cadmium from both sources could be excreted by the fish. The concentration of cadmium within the whole fish increased as the input of cadmium in the diet increased. Zinc, similarly, is available to the flounder both directly from sludge and from zinc accumulated in C. volutator. Only a limited increase in net accumulation of zinc by the flounder was observed upon increased inputs of zinc through the diet, perhaps indicating some regulation of body zinc concentration by the flounder.     

Induction of Cyp1A in Japanese medaka exposed to carbonaceous pitches via water

The objective of these studies was to characterize hepatic Cyp1A induction by complex carbonaceous pitches both in Japanese medaka exposed via aquarium water and in cultured fish liver cells. Carbonaceous pitches were extracted with DMSO and added to aquaria water of medaka or to cultured medaka or trout liver cells, and CyplA induction was assessed by EROD assay. When medaka were exposed to different carbonaceous pitches, EROD activity was induced to different extents, and increased EROD induction was associated with increasing temperature of hydrogenation. EROD induction in cultured fish liver cells was somewhat less sensitive than that observed in vivo. These studies indicate CyplA induction in medaka can be used to detect compounds, such as polyaromatic hydrocarbons, in complex samples and could be used as a biomarker for the presence of these compounds in the environment.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.     

Species and concentrations of selenium and nutrients in Tanabe Bay during red tide due toGymnodinium nagasakiense

A red tide due toGymnodinium nagasakiense was observed in August 1988 in Tanabe Bay, Wakayama Prefecture, Japan. The maximum cell concentration ofG. nagasakiense reached 1×10⁵ cells ml^–1 at the surface water. From May to September 1988, the following were monitored: water temperature, salinity, chlorophylla, D.O., dissolved nutrients (NO₂–N, NO₃–N, NH₄–N, PO₄–P DON, DOP), particulate nutrients (PON, POP) and three dissolved selenium species [Se(IV), Se(VI), Organic Se]. Dissolved inorganic nitrogen (NO₃–N, NH₄–N) decreased but PON, POP, DON, DOP and inorganic phosphate increased at the peak of the bloom. The concentration of organic selenium increased up to the bloom initiation period which started on 5 July, and then the concentration of Se(IV) increased as the concentration of organic selenium decreased at the peak of the bloom (3 August). The strong relationship was found between the concentration of Se(IV) and the cell concentration ofG. nagasakiense (r ²=0.98). The Se(IV) requirement ofG. nagasakiense was 2.89×10^–17 moles cell^–1, which was agreed well with 4.4×10^–17 moles cell^–1 found in a laboratory experiment onG. nagasakiense using selenium spiked artificial sea water medium. The average ratio of Se(IV) to dissolved inorganic nitrogen (DIN) during the red tide bloom was 11441, the ratio of Se(IV) to DIN at the surface with the maximum cell concentration ofG. nagasakiense of 1×10⁵ cells ml^–1 was 1137. These results suggested that selenium may play an important role in red tide outbreak ofG. nagasakiense.