O,p'-DDT induction of vitellogenesis and its inhibition by tamoxifen in Nile tilapia (Oreochromis niloticus)

In order to investigate the mechanism by which o,p'-DDT disrupts endocrine functioning of Nile tilapia in vivo, the estrogenicity of o,p'-DDT was investigated in conjunction with 17beta-estradiol (E2) and tamoxifen. Mature, male tilapia were treated intraperitoneally with o,p'-DDT (60 mg/kg, one dose) or E2 (5 mg/kg, four doses) in the presence or absence of tamoxifen (5 mg/kg, six doses) for 12 days and then plasma vitellogenin (Vtg) (measured as alkaline-labile phosphorous), E2, and testosterone (T) were measured. Vtg levels were increased dramatically by E2 (1,744 +/- 171 microg/ml) and moderately by o,p'-DDT (82 +/- 15 microg/ml) compared with controls (23 +/- 3.5 microg/ml). Tamoxifen alone had no effect on Vtg production, but inhibited both E2 and o,p'-DDT stimulated vitellogenesis. T levels were reduced with E2 administration (1,688 +/- 383 pg/ml) and declined further with the combined treatment of E2 and tamoxifen (281 +/- 70 pg/ml), compared with controls (6,558 +/- 1,438 pg/ml). Tamoxifen or o,p'-DDT alone did not affect T levels, but their combined treatment did (2,069 +/- 647 pg/ml). The results of this study suggest that o,p'-DDT is weakly estrogenic in male tilapia, and that this activity may be mediated through the estrogen receptor.     

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.     

瓦氏黄颡鱼(Pelteobagrus vachelli)卵黄蛋白原的纯化、性质鉴定及ELISA检测方法的建立

腹腔注射17β-雌二醇(E2),使瓦氏黄颡鱼雄鱼在7天内产生卵黄蛋白原(Vtg)。采用凝胶过滤和离子交换两种层析技术,从E2诱导的雄性瓦氏黄颡鱼血浆中分离、纯化出Vtg,采用糖、磷、脂蛋白染色技术证明分离、纯化的蛋白为Vtg,该Vtg在非变性条件下分子量约为240kDa,在SDS变性条件下分子量约为143kDa。纯化的瓦氏黄颡鱼Vtg经检测显示可能含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Vtg,制备了兔抗瓦氏黄颡鱼Vtg多克隆抗血清。用双向免疫扩散法测得抗血清的纯度较高,效价为1︰32;Western blotting检测显示抗血清的特异性较好。以瓦氏黄颡鱼Vtg多克隆抗血清为抗体,以纯化的瓦氏黄颡鱼Vtg为抗原,建立了间接酶联免疫吸附反应(ELISA)方法检测瓦氏黄颡鱼体内Vtg的含量,标准曲线线性部分的线性方程为y=0.099x+0.4529(R2=0.9327),该方法检测的灵敏度为15.6ng/ml,工作范围为31.2—4000ng/ml,在此范围内,标准曲线具有良好的线性。     

Estrogenicity of xenobiotics in rainbow trout (Oncorhynchus mykiss) using in vivo synthesis of vitellogenin as a biomarker

The estrogenicity of several xenobiotics was evaluated using in vivo vitellogenin (Vtg) synthesis in immature rainbow trout as a biomarker. 17β-estradiol, DES and ethinyl estradiol were tested as positive controls. The xenobiotic compounds tested were technical nonylphenol, bisphenol A, butylbenzylphthalate (BBP) and dibutylphthalate (DBF). Measurements of the Vtg concentration was performed with a direct sandwich ELISA. 17β-estradiol, DES and ethinyl estradiol caused up to 100 000-fold increases in the Vtg-levels. Nonylphenol and bisphenol A had the highest estrogenic potency of the xenobiotics increasing the vitellogenin level approximately 300-fold while BBP was only weakly estrogenic, increasing the concentration about 3 times. DPB did not raise the vitellogenin contration above the detection limit.     

Biomarkers for endocrine disruptors in three species of Mediterranean large pelagic fish

The hypothesis that Mediterranean top predator species, such as large pelagic fish, are potentially at risk due to endocrine disrupting chemicals (EDCs), is investigated. The potential estrogenic effects of PHAHs in three fish species of commercial interest, the top predators bluefin tuna (Thunnus thynnus thynnus), swordfish (Xiphias gladius), and Mediterranean spearfish (Tetrapturus belone), were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and mixed function oxidases (EROD, BPMO) as diagnostic tools. High induction of Vtg and Zrp was detected by western blot and ELISA techniques in adult males of X. gladius and T. thynnus thynnus, suggesting that these species are at high toxicological risk in the Mediterranean sea. Comparison of BPMO and EROD activities in the three species indicated, both in male and female, much higher MFO activity in bluefin tuna. This data suggests high exposure of this species to lipophilic xenobiotic contaminants in the Mediterranean environment.     

Estrogenic effect of dioxin-like aryl hydrocarbon receptor (AhR) agonist (PCB congener 126) in salmon hepatocytes

Previous studies have shown that aryl hydrocarbon receptor (AhR) agonists possess anti-estrogenic activities and several mechanisms have been proposed to explain the interactions between AhR and estrogen receptor (ER) signalling pathways. In the present study, we show that 3,3'4,4'5-pentachlorobiphenyl (PCB126 - a dioxin-like AhR agonist) produced estrogenic responses in the absence of ER agonist, in fish in vitro system. We exposed salmon primary hepatocytes to PCB126 (1, 10 and 50 pM) and the ER agonist nonylphenol (NP; 5 and 10 microM) singly and also in combination. Vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) levels were analysed by semi-quantitative ELISA. We observed that the protein levels of Vtg and Zr-proteins were significantly induced in a concentration-specific manner in cells treated with PCB126 and NP, singly or in combination. In general, these results show a novel aspect of dioxin-like PCB effect not previously demonstrated in fish system.     

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.     

中华乌塘鳢雌雄异体和雌雄同体性类固醇激素含量的季节变化与差异

采用酶联免疫吸附法测定了野生中华鸟塘鳢（Bostrichthyssinensis）不同季节血清E2和1l—KT含量的变化,并比较了生殖季节养殖雌雄同体与野生雌雄异体之间E,、11一KT和17仪,2013一P三种性类固醇激素含量的差异．结果表明,野生雌鱼和雄鱼血清E：和11一KT的含量均具有明显的季节变化,其高低均依次为5〉8〉2〉11月．在相同月份,雌鱼的E,含量显著高于雄鱼,而雄鱼的ll—KT含量显著高于雌鱼．生殖高峰期,雌雄同体E：含量高于雌雄异体雄鱼,但低于雌雄异体雌鱼;雌雄同体l1一KT和17仅,20]3一P含量均高于雌雄异体雌鱼和雄鱼;野生雌鱼和雄鱼血清170L,2013一P含量没有显著差异．性类固醇激素在中华鸟塘鳢的生殖过程中起着重要作用,E：是野生中华乌塘鳢雌鱼的主要雌激素,而11一KT则是野生雄鱼的主要雄激素．     

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius.

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.     

Evaluation of ecotoxicological effects of endocrine disrupters during a four-year survey of the Mediterranean population of swordfish (Xiphias gladius)

In this project we investigated the ecotoxicological effects of endocrine disrupters in a four-year survey of the Mediterranean population of swordfish (Xiphias gladius). In the Mediterranean environment, top predators, such as swordfish, accumulate high concentrations of polyhalogenated aromatic hydrocarbons (PHAHs) and toxic metals, potentially incurring high toxicological risk. The effects of organochlorines and trace elements (Hg, Cd and Pb) in 192 swordfish specimens, caught in the Strait of Messina, Sicily, Italy, were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and CYP1A (BPMO, EROD) activities. Vtg and Zrp were found to be dramatically induced in some adult male specimens, suggesting that this species is highly exposed to estrogens in the Mediterranean Sea. A role of organochlorines in this induction phenomenon is suggested by the statistically significant correlations between Zrp in plasma and PCB concentrations in muscle (p<0.032) and Vtg in plasma and PCB concentrations in liver (p<0.034) of male specimens. Levels of trace elements in liver were in the following ranges: Hg 1-22, Cd 1-28 and Pb 0-1.6 ppm d.w. These data indicate potential reproductive alterations in large pelagic fish and suggest the need for continuous monitoring to avoid reductions in the population of this fish species of high commercial and ecological interest.     

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.     

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

Several genes normally induced by estradiol (E(2)) 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 E(2) 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 E(2) 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.     

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.)

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.     

Fish Zona radiata (eggshell) proteins: evaluation and validation as a biomarker for xenoestrogen monitoring

New cellular and molecular tools are enabling researchers to study the basic mechanisms of action of natural reproductive hormones, and of endocrine disrupters, including xenoestrogens. Zonagenesis is defined by the hepatic synthesis of the eggshell Zona radiata proteins (Zrp), their secretion and transport in the blood and their uptake by maturing oocytes. Zrps are comprised of three-to-four protein monomers in the 50–60 kDa MW range and show structural similarity to proteins of the Zona pellucida; an extracellular envelope that lies immediately outside the plasma membrane in the eggs of placental mammals. We studied the xenoestrogen-induced elevation in hepatic mRNA and plasma levels of Zrp and vitellogenin (Vtg) in several fish species. Eggshell Zrp protein and mRNA was induced in juvenile salmon by exposure to nonylphenol (NP: given singly or in combination with other xenoestrogens/xenobiotics). In English sole (Pleuronectes vetulus), Zrp was induced in the plasma of mature female, E₂- and NP-treated male fish. We are currently using salmon cDNA probes to study estrogen-receptor, Zrp and Vtg mRNA expression in English sole treated with E₂- and NP. Validation in a semi-field experiment showed that effluent from an oil refinery treatment plant caused a dose-dependent induction of Zrp and Vtg in juvenile salmon. However, a component corresponding to Zrp-β was more responsive than Zrp-α and Zrp-γ and also more than Vtg. Zrp seem to be a sensitive complement to Vtg analysis, and appear to be a more ecologically significant biomarker of estrogenicity.     

o,p′-DDT induction of vitellogenesis and its inhibition by tamoxifen in Nile tilapia (Oreochromis niloticus)

In order to investigate the mechanism by which o,p′-DDT disrupts endocrine functioning of Nile tilapia in vivo, the estrogenicity of o,p′-DDT was investigated in conjunction with 17β-estradiol (E₂) and tamoxifen. Mature, male tilapia were treated intraperitoneally with o,p′-DDT (60 mg/kg, one dose) or E₂ (5 mg/kg, four doses) in the presence or absence of tamoxifen (5 mg/kg, six doses) for 12 days and then plasma vitellogenin (Vtg) (measured as alkaline-labile phosphorous), E₂, and testosterone (T) were measured. Vtg levels were increased dramatically by E₂ (1744±171 μg/ml) and moderately by o,p′-DDT (82±15 μg/ml) compared with controls (23±3.5 μg/ml). Tamoxifen alone had no effect on Vtg production, but inhibited both E₂ and o,p′-DDT stimulated vitellogenesis. T levels were reduced with E₂ administration (1688±383 pg/ml) and declined further with the combined treatment of E₂ and tamoxifen (281±70 pg/ml), compared with controls (6558±1438 pg/ml). Tamoxifen or o,p′-DDT alone did not affect T levels, but their combined treatment did (2069±647 pg/ml). The results of this study suggest that o,p′-DDT is weakly estrogenic in male tilapia, and that this activity may be mediated through the estrogen receptor.     

The correlation between bioaccumulation and pattern of stress-related genes expression of black sea bream (<Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>) by cadmium exposure

In order to correlate the expression of detoxifying enzyme genes and Cd accumulation in black sea bream, we analyzed four tissues (brain, gills, liver, and muscle) from black sea breams that were exposed to four different concentrations of Cd (0, 2, 13, and 25 mg/L) for various durations (0, 24, 48, 72, and 96 h). The highest level of Cd was accumulated in the liver, followed by the gills, brain, and muscle. The accumulation of Cd was significantly correlated with the duration of exposure and the concentration in brain, gill, and liver tissue, but not in muscle tissue, and the rate of accumulation increased with Cd concentration. The expression of metallothionein II (MT II) mRNA exhibited a similar pattern as Cd accumulation, especially in that the expression of MT II mRNA decreased in muscle tissue with increases in exposure duration. In contrast, the expression of cytochrome P450 1A (CYP1A) mRNA was highest in the liver, followed by brain, muscle, and gill tissues, and in gills and muscle tissue of Cd-exposed fish, the expression of CYP1A mRNA fell below that of the control fish. Overall, the liver of black sea bream was the most sensitive to Cd exposure, and the expression of MT II mRNA was 200-fold greater than the control fish. These findings indicate that the detoxification mechanisms of black sea bream are influenced by both MT II and CYP1A and that the genes participate in the detoxification of different tissues.     

Array technology as a tool to monitor exposure of fish to xenoestrogens

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).     

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and Octa-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days 1 and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n=5 for each group) and 14 (n=6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYP1A were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17β for Vtg and Zrp expression, and β-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.     

Parentage determination of black sea bream(Acanthopagrus schlegelii) for stock enhancement:effectiveness and loss of genetic variation

The stock enhancement programs for black sea bream Acanthopagrus schlegelii have been conducted in China for a fewyears.However,little information has been reported concerning the effectiveness and genetic effect of black sea bream stock enhancement.In order to detect the contribution of released individuals in Zhujiang River Estuary(ZRE) and Daya Bay(DB),six microsatellite markers were used to identify the hatchery-released individuals.In addition,this pedigree of hatchery populations(broodfish and hatchery-released offspring) was traced to detect the number of effective parents(N_e),the inbreeding coefficient and the decrease of genetic variability in the reproduction.The pedigree reconstruction showed that at least 69(out of 93) broodfish had offspring.The estimated N_e was 54.8,consequently the inbreeding coefficient was 0.91%.The genetic diversity of hatchery-released offspring was lower than that in that of broodfish(heterozygosity alleles,0.727-0.774),some alleles(number of alleles,61-69) and genetic variance were lost during reproduction.It was observed that wild samples had higher levels of genetic diversity compared with hatchery populations as well as recaptured samples in releasing area.A total of 128 hatchery-released black sea bream were identified among 487 recaptured samples in ZRE,while a total of 15 samples were identified among 96 samples in DB.In summary,there was a high survival of released fish.Nevertheless,the results provided evidence to consider a loss of genetic variation in hatcheryreleased stock and a negative genetic effect of the stock enhancement.