Oral single pulse exposure of flounder Platichthys flesus to 4-tert-octylphenol: Relations between tissue levels and estrogenic effects

The accumulation of 4-tert-octylphenol and the associated estrogenic effects were studied after a single pulse exposure to flounder Platichthys flesus. 4-tert-octylphenol was administered orally in a single dose of 50 mg kg(-1) and tissue (liver, muscle and testis) and plasma concentrations of 4-tert-octylphenol as well as plasma vitellogenin were measured 3, 6, 12, 18, 24, 48, 72, 144 and 216 h after administration of the dose. Concentrations of 4-tert-octylphenol in plasma and tissues were determined by Liquid Chromatography Mass Spectrometry (LC-MS). 4-tert-octylphenol was detectable in liver, testis, muscle and plasma 3h post administration and an accumulation was observed in liver, muscle and plasma up to 12 h and in testis 18 h post administration, respectively. The maximum concentrations of 4-tert-octylphenol in liver, muscle and testis were 67, 3.2 and 6.8 microg g(-1), respectively. An increase in plasma vitellogenin levels was seen 48 h post administration and the vitellogenin level continued to increase (from <100 ng ml(-1) to 1.4 mg ml(-1)) until the end of the experiment 9 days after the administration of 4-tert-octylphenol.     

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.     

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.     

Effects of prolonged exposure to 4-tert-octylphenol on toxicity and indices of oestrogenic exposure in the sand goby (Pomatoschistus minutus, Pallas)

Alkylphenolic compounds are present in estuarine and marine environments. They are moderately bioaccumulative, and oestrogenic to fish following three week, in vivo, exposures. The effects of prolonged exposure are, as yet, unclear. Sand goby (Pomatoschistus minutus) were exposed to measured concentrations of 4-tert-octylphenol (OP) in a 28-day, dose-ranging study, and a six month temporal response study. Following 28-days exposure to measured 31+/-6 or 101+/-47 microg l(-1) OP (mean+/-SD), immature male sand goby showed elevated vitellogenin (VTG) mRNA expression. Plasma alkali-labile phosphate concentrations were elevated in both sexes and were higher in females than males. Although measured OP concentrations were variable, they were significantly different for each treatment and a range of concentration-dependent effects were observed. The LC(50) for chronic OP exposure (8 weeks) was 29+/-6 microg l(-1), and both sexes demonstrated concentration and duration dependent increases in VTG mRNA expression. Exposure to 28+/-20 microg l(-1) OP (median+/-95% CI) for 6 months inhibited development of male nuptial colouration and sperm duct glands. These findings are discussed in relation to environmental concentrations of alkylphenols and ecological concerns.     

The assessment of vitellogenin as a biomarker of exposure to estrogenic compounds in two Australian perciformes

Vitellogenin (Vtg) is a yolk protein precursor that has been identified as a sensitive biomarker for exposure to estrogenic compounds. We evaluated specific monoclonal and polyclonal antibodies for reactivity with plasma Vtg from two Australian Perciformes, the tropical barramundi (Lates calcarifer) and the temperate black bream (Acanthopagrus butcheri). Blood plasma from 17beta-estradiol exposed (E2) male barramundi (20 mg kg(-1)) and male black bream (2.5-5.0 mg kg(-1)) were sent to Biosense Laboratories (Norway) for cross-reactivity testing using their extensive anti-Vtg antibody selection. Indirect ELISA results determined barramundi plasma displayed the highest binding affinities to ND-3G2 (monoclonal-Mab) and PO-1 (polyclonal-Pab). Black bream was most cross-reactive with ND-1C8 (Mab) and PO-2 (Pab). Next, plasma was assessed for Vtg induction in E2-dosed (5 mg kg(-1)), hatchery-reared barramundi and black bream versus a non-injected control group. Vtg production was assessed by Western blot and indirect ELISA using ND-3G2 and ND-1C8 Mabs, respectively. A prominent band was identified in the range of 100-200 kDa for all female black bream and for all E2-treated barramundi and black bream males, which was confirmed as Vtg by Western blot. Indirect ELISA results for barramundi demonstrated highly significant differences in E2-dosed fish as compared to control fish (Student t, P<0.001). E2 male black bream were significantly different than control males (Student t, P<0.001) and control and E2 females displayed highly significant differences (Student t, P<0.001). These results indicate that exposure to 17beta-estradiol induces significant Vtg production in males of the two Australian Perciformes, with potential use as a biomarker for exposure to estrogenic compounds.     

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 cta-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 I 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 CYPIA 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-17beta for Vtg and Zrp expression, and beta-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.     

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.     

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: an issue for design and interpretation of monitoring and research programmes

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.     

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.     

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.     

Vitellogenin as biomarker for estrogenicity in flounder Platichthys flesus in the field and exposed to 17α-ethinylestradiol via food and water in the laboratory

The ability of 17α-ethinylestradiol (EE2) to elevate vitellogenin levels were investigated in male flounder Platichthys flesus and vitellogenin concentrations in flounders from the Danish coastal environment were determined. Male flounders were exposed to 17α-ethinylestradiol (EE2) via food or water. Average vitellogenin concentrations in the control fish ranged between 25 and 100 ng mL⁻¹. Exposure to 5.1, 8.1 and 16.8 ng EE2 L⁻¹ in water and 500 and 5000 ng EE2 kg⁻¹ body weight (bw) every second day in the food increased the plasma vitellogenin concentration in a concentration and time dependent manner, whereas exposure to 2.7 ng EE2 L⁻¹ in water for 21 d and 5 and 50 ng EE2 kg⁻¹ bw for 12 days in the food did not. EE2 could be detected in liver and testes (but not in muscle) after exposure to 8.1 and 16.8 ng EE2 L⁻¹ in the water and 5000 ng EE2 kg⁻¹ bw in the food; the highest concentration was 6 ng g⁻¹ wet weight in liver. The majority of the male flounders collected from nine coastal Danish sites from 1999 to 2004 had vitellogenin concentrations below 100 ng mL⁻¹, and only at two sites moderate estrogenic inputs were indicated.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

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.     

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.     

Pathology of the liver, kidney and gonad of flounder (Platichthys flesus) from a UK estuary impacted by endocrine disrupting chemicals.

The Mersey estuary is highly contaminated with xenobiotics compared to the nearby Dee estuary. Male flounder, a migratory flatfish caught in the Mersey frequently contains high blood concentrations of the oestrogen controlled, female protein vitellogenin, suggesting that Mersey flounder have been exposed to endocrine disrupting contaminants. Males caught from the Dee contain lower blood vitellogenin levels. Preliminary histopathological examination of 410 flounder from both estuaries, focuses on the liver, kidney and gonads. Hepatic tubular vacuolation, foci of cellular alteration, and hepatocellular tumours were seen. Renal and gonadal pathology included the presence of two Mersey fish with enlarged and abnormal glomeruli, three phenotypic male Mersey flounder with unilateral intersex gonads and one male with bilateral, abnormal ovarian follicular components in the testis. These initial results provide pathological evidence of xenobiotic exposure in flounder sampled from both estuaries. Preliminary findings from flounder sampled from Millport, an offshore site in Southwest Scotland, showed no histopathological evidence of xenobiotic exposure.     

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。     

Isolation of differentially expressed genes from contaminant exposed European flounder by suppressive, subtractive hybridisation

Commercially farmed European flounder (Platichthys flesus) were treated with benzo[a]pyrene or cadmium under controlled laboratory conditions. In addition, animals were collected from the polluted Tyne and relatively clean Alde estuaries in the UK. Potential differentially expressed genes were isolated from liver tissue by means of suppressive, subtractive hybridisation. A total of 284 different mRNAs were identified as having the potential for being differentially expressed, of which 84 could be tentatively identified by homology with database sequences. These clones will form part of a flounder cDNA array for validation and future applications, with full identification being pursued for those expressed sequence tags for which differential expression can be confirmed by array technology.     

Increased potential for NAD(P)H-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants.

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51-160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg-1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake J?rnsj?n compared to clean Lake V?nern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.     

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.     

Effect of dietary copper exposure on accumulation, growth and hematological parameters of the juvenile rockfish, Sebastes schlegeli

Copper accumulation, and its effect on growth and hematological parameters were investigated in juvenile rockfish, Sebastes schlegeli (mean length 11.83+/-0.03 cm, and mean weight 26.02+/-0.23 g), after sub-chronic dietary Cu exposure (0, 50, 125, 250 and 500 mg/kg) for 60 days. The profile of Cu accumulation among tissues in rockfish is dependent on the exposure periods and Cu concentration. Liver of rockfish is a more important storage tissue than other tissues, and the order of Cu accumulation in tissues was liver > intestine > kidney > gill > muscle. Cu reduced the growth rate, and there was an inverse relationship between growth and Cu concentration (> 50 mg/kg). The RNA:DNA ratios were not affected by exposure and there was no correlation between growth rate and RNA:DNA ratio in the liver and muscle. There was no significant effect of exposure on blood parameters except for magnesium. Cu exposure increased GOT and GPT serum concentrations with increasing time and dose.