Estradiol and estriol suppress CYP1A expression in rainbow trout primary hepatocytes

Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17beta-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 10(-7) M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish.     

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.     

A comparative study of the differential blood cell counts of winter flounder (Pseudopleuronectes americanus) collected in New England

Major alterations in the distribution of peripheral blood cells were observed among winter flounder (Pseudopleuronectes americanus) from Martha's Vineyard (MV), East Cape Cod Bay (ECCB), Fox Island (FI) (Narragansett Bay), Central Long Island Sound (CLIS), Gaspee Point (GP) (Narragansett Bay), Black Rock Harbor (BRH) (Bridgeport, CT), New Bedford Harbor (NBH) and Quincy Bay (QB) (Boston Harbor). Collection locations ranged from relatively uncontaminated offshore (MV) and nearshore (ECCB) sites to the more contaminated estuarine (FI, CLIS) and urban embayments (GP, BRH, NBH, QB). Differential diagnosis demonstrated a shift in lymphocyte counts from 28% (MV) to 54% (QB) that increased with the incidence of liver lesions in relation to known and suspected levels of sediment chemical contamination. A significant change in the ratio of circulating immature to mature erythrocytes was observed in flounder collected from Boston Harbor. Immature erythrocytes comprised 18% of the total erythrocyte population of fish from QB in comparison to 6% observed in flounder from ECCB.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E₂) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E₂ and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E₂ and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

外源激素对养殖牙鲆血浆睾酮和雌二醇含量的影响研究

雄性牙鲆单独注射促黄体激素释放激素类似物(LHRH-A)(每千克质量的注射量,下同)25μg/kg或人绒毛膜促性腺激素(hCG)1 000 IU/kg,血浆睾酮(T)含量在注射后6 h大幅度降低,12h时达到最低值,以后逐渐回升;LHRH-A与hCG联合注射[LHRH-A(12.5μg/kg)+hCG(500 IU/kg)],注射后6 h血浆T含量剧烈下降,24 h时达到最低点,以后缓慢回升;对照组雄鱼血浆T含量随着采样进行而呈现降低趋势.雌鱼单独LHRH-A后,血浆雌二醇(E₂)水平逐渐下降,24 h时达到最低值,30 h后略有回升;单独注射hCG后,血浆E₂含量缓慢下降,30 h达到最低值;LHRH-A与hCG联合注射,注射后6 h血浆E2含量逐渐下降,24 h时达到最低点,30 h时缓慢回升.对照组雌鱼血浆E₂水平也随着采样的进行而逐渐降低.结果表明:单独或联合注射LHRH-A和hCG降低了雌、雄牙鲆血浆E₂和T含量,与对照组结果相同.     

Size at first maturity of two species of flounders in the Hauraki gulf,New Zealand

The length at first maturity of the sand flounder, Rhombosolea plebeia (Richardson), and the yellow‐belly flounder, Rhombosolea leporina Gunther, (Teleostei: Heterosomata) in the Hauraki Gulf was investigated by the incidence of sexually tnature and immature fish grouped at 1‐cm length intervals. All male sand flounders and yellow‐bellies were sexually mature at sizes considerably less than the legal minima of 22.9 cm (9 in.) and 25.4 cm (10 in.) respectively. About 95% of female sand flounders and about 15% of female yellow‐bellies matured before reaching the respective minimum legal sizes. Most (i.e., over 50%) female sand flounders and yellow‐bellies were mature when they reached lengths of 18 cm and 26 cm respectively.     

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.     

Spawning and fecundity of two flounder species in the Hauraki gulf,New Zealand

Spawning grounds and spawning times of the sand flounder, Rhombosolea plebeia (Richardson), and the yellow‐belly flounder, Rhombosolea leporina Gunther, in the Hauraki Gulf are described. The occurrence of female fish at different stages of ovarian development at different stations during the year, the distribution of eggs in the plankton, and changes in ovary weight during the year were used as indicators of spawning grounds and spawning times.

The sand flounder was found to spawn in the waters to the east of Waiheke and Ponui Islands, at the northern end of the Firth of Thames, from June to November.

Yellow‐belly flounders spawned during September, October, and November, slightly to the south of the sand flounder spawning grounds in a belt extending from Tapu, on the eastern side of the Firth of Thames, north‐westwards towards Ponui Island.

Fecundity of both species was approximately proportional to the weight of the fish, or to the length cubed, and less than proportional to the ovary weight. The mean fecundity of sand flounders in the Hauraki Gulf varied from approximately 100,000 eggs in a fish of 18 cm to 500,000 in a 30 cm fish. That of yellow‐belly flounders varied from approximately 250,000 eggs in a fish of 30 cm to 1.25 million in a 45 cm fish.     

Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.     

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.     

不同盐度与雌二醇投喂对花鲈幼鱼生长性能的影响

为了探寻不同盐度和17β-雌二醇(E2)对花鲈(Lateolabrax maculatus)幼鱼生长性能影响,作者设置3个实验盐度梯度,分别为正常养殖盐度(30)、低盐度(0)、高盐度(45),对初始平均体质量分别为0.10607、0.84341、10.94173 g/尾的花鲈,急性盐度胁迫后进行养殖实验,并对前两批幼鱼进行E2投喂处理。结果表明,在3种规格盐度实验处理中,低盐组鱼类都表现出比较显著的生长优势(P0.05),随着规格增大,淡化所需的时间越短,在盐度处理组中,高盐处理组与正常组鱼类平均体质量在各阶段中无显著差异(P0.05)。经50与25 mg/kg剂量的E2投喂处理后,处理组花鲈幼鱼在体长、体质量方面都出现了明显抑制,且肝质量指数显著增加并出现腹水现象,同时,高盐处理与雌二醇投喂之间可能存在协同作用,加强了对花鲈幼鱼早期生长的抑制作用。目前对花鲈幼鱼淡化过程中恢复情况的研究较少,同时未见有关E2投喂对花鲈幼鱼生长性能影响的研究,作者研究结果对北方花鲈在不同盐度水体中的养殖模式推广提供科学依据,并对E2诱导性别分化同时所产生的生长性能降低的风险评估提供参考。     

Time-course studies of the biotransformation enzymes in control rainbow trout when adjusting to new habitats

Hepatic monooxygenase enzyme activities and relative cytochrome P4501A protein content were measured to evaluate the time-course alterations in rainbow trout after change in living habitat. Fish were transferred from one fish farm to the tanks of another hatchery and/or into cages kept in a lake. In the new habitats, cytochrome P450-dependent enzyme activities in rainbow trout decreased, and were at their lowest levels after two or three weeks in the summer. Later the activities partly reversed. The immunodetection of cytochrome P4501A protein expressed a similar trend as for catalytic monooxygenase activities.     

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.     

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.     

Using cytochrome P450 to monitor the aquatic environment: Initial results from regional and national surveys

We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAA's Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.     

Biotransformation enzyme activities and PCDD/PCDF levels in pike caught in a Swedish Lake

Recently, analysis of organochlorines in sediment and in pike from Lake Vänern, Sweden, showed a north-south gradient of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). In the present study, good correlations were found between muscle 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents and liver ethoxyresorufin-O-deethylase (EROD) activities (and P4501A levels) in pike caught at three sampling sites along the north-south gradient in the lake. This comparison between tissue levels of PCDD/PCDF and EROD activity (and P4501A) may indicate that TCDD or structural analogs could be responsible for the observed induction of the pike P450 system. It must, however, be taken into account that the analysed contaminants often do not appear alone but are, rather, accompanied by a range of other substances which may be inducers or antagonists.     

人工诱导雌核发育牙鲆的染色体及核型证明

于1994-1996年,分别在威海海洋渔业捕捞公司（石岛）和鸿洋实业总公司龙须岛育苗场采集人工培育的3－5龄牙鲆亲鱼,采用紫外线照射法使牙鲆精子遗传物质失活,并用冷休克法抑制受精卵第二极体释放,从而获得雌核发育二倍体牙好。原肠期采用空气干燥法、Giemsa染色,得到雌核发育二倍体、正常二倍体及单倍体的染色体制片,进行染色体和核型的分析。结果表明,牙鲆的雌核发育二倍体和正常二倍体的染色体数均为2n=48,核型为48t,即48条端部着色点染色体,臂数NF=48,两者的核型设有明显差异;单倍体为24条端部着丝点染色体;在3个组别中,第一号染色体上都有一明显的次缢痕。雌核发育二倍体牙鲆的诱导率为98％。     

Flounder growth and production as indicators of the nursery value of marsh habitats in a Mediterranean lagoon

Estuarine marshes are known as suitable nursery areas for many marine migrant fishes, such as flounder. The potential nursery value of such habitats was investigated in the Venice lagoon, by using growth and production of 0-group flounder as indicators. Size–frequency distribution analysis was performed on fish samples collected fortnightly, from March 2004 to June 2005, in two marsh sites, Dese and Tessera, differing in their origin and environmental conditions. Samples were mostly composed of juvenile individuals, belonging to 0- and 1-group cohorts (the latter being present in Tessera only). A higher total production, either annual or monthly, and faster growth of 0-group flounder was observed in Dese, associated to a higher ecological performance of 0-group individuals in this site, as indicated by the higher P:B ratio values. Dese is a site located in a marsh complex characterized by the relevant influence of a nearby river, and the observed higher potential nursery value of this marsh area with respect to the other is discussed in the light of the higher trophic status and other environmental conditions in this site. The production results were also compared to those from other estuarine environments commonly acknowledged as important nurseries for European flounder.