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.     

Estradiol-17β and o,p′-DDT stimulate gonadotropin release in Atlantic croaker

Xenobiotic estrogens have the potential to act at a variety of estrogen-responsive target tissues on the hypothalamic-pituitary-gonadal axis. However, to date most studies in fish have focused on stimulation of vitellogenin synthesis by the liver. In the present study the effects of the xenoestrogen o,p′-DDT and estradiol-17β on the neuroendocrine control of gonadotropin secretion were compared. Atlantic croaker (Micropogonias undulatus) were exposed to o,p′-DDT (0.02 and 0.1 μgg⁻¹ body weight day⁻¹) in the diet for 3 and 7 weeks during the gonadal recrudescence phase. The o,p′-DDT exposure elicited a significant increase in plasma gonadotropin levels after both 3 and 7 weeks of exposure. The stimulatory effect of o,p′-DDT on basal (spontaneous) gonadotropin release was accompanied by a slight increase in ovarian growth as evidenced by the increase in gonadosomatic index. It appears that the stimulation of gonadotropin release by o,p′-DDT during early-recrudescence phase results in enhanced ovarian growth. A comparable stimulatory effect was observed with estradiol-17β treatment during early- and late-recrudescence phases of the ovarian cycle using three injections on alternate days and slow release silastic implants (five days). The present study provides the first evidence for an estrogen-like action of o,p′-DDT on gonadotropin release in a teleost model.     

Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitellogenic rainbow trout

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre⁻¹ for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.     

Tributyltin and triphenyltin induce spermatogenesis in ovary of female abalone,Haliotis gigantea

Two-month flow-through exposure experiments of tributyltin (TBT) and triphenyltin (TPhT) were conducted with abalone, Haliotis gigantea. Nominal concentrations of 100 ng TBT/l and 100 ng TPhT/l caused significant spermatogenesis in ovaries of exposed females. There were also significantly more contracted primary oocytes observed in females exposed to either TBT or TPhT than controls. The incidence of two types of unknown cells was also significant in females exposed to TPhT. No significant histological changes were observed in testis of exposed males. This ovarian spermatogenesis caused by TBT and/or TPhT resembles gastropod imposex. Remarkably high concentrations of TBT and TPhT were observed in the head (including central nervous system ganglia), compared to muscles concentrations. Accumulation of TBT and TPhT in the head may disturb reproductive hormonal regulators through neuropeptides released from ganglia. This, as well as possible aromatase inhibition, may be one of the inducers for spermatogenesis in the abalone ovaries.     

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.     

Comparative vitellogenic responses in three teleost species: extrapolation to in situ field studies.

Induction of vitellogenin (VTG) was compared among three teleostean species to determine their relative sensitivity of exposure to 17 beta-estradiol (E2). Japanese medaka (Oryzias latipes), sunshine bass (Morone saxatalis x Morone chrysops) and channel catfish (Ictalurus punctatus) were exposed to aqueous concentrations of E2 ranging from 10 to 100,000 ng/l for 21 days. Respective EC50 values for plasma VTG detected by western blot in medaka, catfish and bass were 200, 170 and 1560 ng E2/l. Since these EC50 values are based on VTG induction curves calculated relative to control values, they indicate differences in species' sensitivity to E2 exposure. Catfish and bass VTG responses obtained in laboratory exposures were compared to VTG responses previously observed with 21-day wastewater treatment plant effluent exposures. Plasma VTG induction in effluent-exposed fish ranged from 14 to 82% above reference values depending on species. Extrapolation of field responses with laboratory-exposed fish indicate catfish and bass were exposed to the equivalent of 27-240 ng E2/l in sewage effluent.     

Immune function, hepatic CYP1A, and reproductive biomarker responses in the gulf killifish, Fundulus grandis, during dietary exposures to endocrine disrupters.

The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses. We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively. We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period. The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals. The contaminated diet suppressed secondary antibody responses, but only in the morning. The contaminated diet also altered CYP1A, but not vitellogenesis. In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction. Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females. This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.     

Environmental estrogens: dose–response relationships for vitellogenin formation and reproductive toxicity in male rainbow trout

The appearance of the egg-yolk protein vitellogenin (Vg) in plasma of male fish is a sensitive indicator of exposure to estrogenic compounds. We have been studying the kinetics of Vg formation and excretion in rainbow trout with a goal towards developing an integrated pharmacokinetic–pharmcodynamic (PK–PD) model to quantitatively relate cumulative estrogenic exposure of fish to the expression and appearance of Vg in plasma. We administered graded doses of ethynylestradiol (EE2), o,p-DDT, DDD and DDE and octylphenol to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of the xenobiotics and Vg were simultaneously quantified using ELISA and GC–MS or GC–ECD. In separate experiments, sexually mature trout were exposed to graded water concentrations of EE2 for 3 months and various parameters indicative of the functional status of the male reproductive system determined. These parameters included tissue-somatic indices, histopathological evaluation, spermatocrit, sperm motility (quantified using computer-assisted-motion analysis) and viability of semen based on fertilization assays using eggs harvested from untreated trout. Results from fertilization assays indicated that 12 week exposure to EE2 concentrations of 10 and 100 ng/l caused a 50% reduction in the fertilization rate of semen harvested from exposed trout. PK–PD modeling strategies proved valuable tools for linking chemical exposures to Vg formation.     

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss)

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid). Male trout were orally exposed for 17 days to 55 microg/kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L. After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.     

Transport and accumulation of organochlorines in the ovaries of Atlantic croaker (Micropogonias undulatus)

The role of vitellogenin in the transport of organochlorines was investigated in Atlantic croaker (Micropogonias undulatus) by exposing them through the diet to o,p′-DDT at a concentration of 10.8 μg/100 g fish/day for 14 days or Aroclor 1254 (PCB) at a concentration of 0.5 mg/100 g fish/day for 30 days during gonadal recrudescence. Tissue samples were taken from the fish at various times after initial exposure, and o,p′-DDT and PCBs were extracted in acetonitrile and analyzed by gas chromatography. Analysis of the ovarian tissue collected 2 and 3 weeks from the start of exposure revealed that the o,p′-DDT concentration increases as the gonadosomatic index (GSI) increases (r² = 0.63), with accumulation ranging from less than 1% to as much as 8% of the total dosage. Interestingly, o,p′-DDT did not accumulate in the testes during the same exposure period. Accumulation of PCBs was found to be 40 times higher in the ovaries than in the testes. Gel filtration of plasma from exposed females showed that o,p′-DDT elutes in the low density lipoprotein and vitellogenin fractions. Control plasma incubated with o,p′-DDT at 4 °C for 16 h followed by chromatography on Sepharose 6B gave similar results with an o,p′-DDT concentration of 0.6μg/mg protein in the vitellogenin fraction. Furthermore, both o,p′-DDT and PCBs were found to bind to purified croaker vitellogenin. These results suggest that lipoproteins, including vitellogenin, are involved in the transport and accumulation of organochlorines in the ovaries of exposed fish.     

Uptake of estradiol from sediment by hornyhead turbot (Pleuronichthys verticalis) and effects on oxidative DNA damage in male gonads

Male and female hornyhead turbot (Pleuronichthys verticalis) were exposed to four concentrations (0, 0.75, 14.7 and 46.5 ng/g dry weight) of E2-amended sediment for 7 days. Sediment-derived E2 was bioavailable to the flatfish, though the route of uptake was unclear. A concentration of 46.5 ng/g E2 in sediment led to a significant increase in vitellogenin concentrations in the plasma in both sexes after 7 days of exposure. Though plasma E2 concentrations increased significantly in males at sediment E2 concentrations of 0.75 ng/g dry weight and above, a dose-dependent increase was not observed. There was also no correlation between sediment E2 concentrations, plasma E2 concentrations, and oxidative DNA damage in male gonads. The results suggest that the DNA damage previously seen in the gonads of feral hornyhead turbot at a sewage outfall is likely not caused by acute exposure to exogenous E2 from sediments.     

Influence of exposure history on vitellogenin induction in medaka: a case of ‘estrogen memory’?

In this study, we exposed female medaka to aqueous solutions of o,p′-DDT until tissues residues reached approximately 100 g/g. Male offspring of exposed females and unexposed males that were raised to 6 months of age differed from untreated controls in their response to subsequent 4-day static exposure to 17-β-estradiol (E2; 12 μg/l as determined by steroid EIA). Homogenized liver samples from males from DDT treatments, females from DDT treatments, control males and control females produced two vitellogenin bands on a Western blot with no qualitative differences in band size and location between the groups. To quantify the relative level of vitellogenin, 5 μg of protein from each sample was analyzed by slot blot. A statistical comparison revealed a significant difference in optical density of the slots between control males and DDT exposed males (analysis of variance, n=18, P=0.021; Tukey test, n=9, P=0.047) indicating that previous transgenerational exposure to DDT enhanced the vitellogenic response to E2 exposure in males. These results indicate that exposure to estrogenic chemicals may produce an ‘estrogen memory’ which potentiates the response to a subsequent exposure event.     

Toxicokinetics of heavy metals from a mining spill using Carcinus maenas

The knowledge of the reproduction and growth background related to the shore crab Carcinus maenas promotes the use of this crab as a model crustacean to asses the potential for endocrine disruption in crustaceans. In addition, an enzyme linked inmunosorbent assay (ELISA), sensitive to the shore crab vitellogenin in serial hemolymph samples allows determination of the extent of disruption of the process of vitellogenesis in female crabs and its likely impact on reproductive output. Intermoult females Carcinus maenas were exposed to concentrations of Cd: 3 microgl(-1), Cu: 15 microgl(-1) and Zn: 700 microgl(-1) determined at the Guadalquivir estuary after the Aznalcóllar mining spill, during 21 days. Crab hemolymph samples, were taken every seven days, and analyzed through an ELISA for Carcinus maenas vitellogenin. Vitellogenin concentration along the time was fitted to a first order kinetic approach. Results showed a good correlation among experimental values and estimated ones. Metal exposure resulted in an increase in vitellogenin concentration in hemolymph, especially for cadmium.     

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.     

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.     

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.     

DDT对细巧华哲水蚤（Sinocalanus tenellus）摄食、呼吸、存活和生殖的影响研究

DDT(dichloro-diphenyl-trichloroethane) as a type of organochlorine pesticides, is an important component of pesticides pollution whose impact on the marine ecosystem is urgently to be evaluated. To investigate the biological effects of DDT on the marine ecosystem, copepods being the main contributor of secondary productivity in the marine ecosystem, were selected as target animals. The influence of DDT on the feeding, respiration, survival, and reproduction of Sinocalanus tenellus(S. tenellus) was analyzed and the antioxidant enzymes activities in the individuals were measured under different exposure concentrations of DDT. The 48 h median lethal concentration(LC50) and 96 h LC50 of DDT to S. tenellus were 5.44 and 2.50 μg/dm3, respectively. The filtration rates, grazing rates, and respiration of S. tenellus decreased apparently with increased DDT concentrations. Under lower concentration(625 ng/dm3) of DDT, the activities of the antioxidant enzymes, including superoxide dismutase and catalase in the animals increased significantly compared with those in the animals without any exposure to DDT, which suggested that the antioxidant enzymes can protect the animals from oxidative damage. However, the activity of the antioxidant enzyme decreased when the animals were exposed to higher concentration(1 250–2 500 ng/dm3) of DDT. The survival rate of both females and males was reduced when they were exposed to DDT less than 250 ng/dm3, but females showed higher survival rate than males when they are under the same concentration. The hatching ratio and the egg diameters of S. tenellus decreased significantly when they were exposed to DDT with a concentration of 25 and 250 ng/dm3, however, the cumulative egg production did not show any significant variation when the animals were exposed to the above DDT concentration. These data in the preset study suggested that exposure to DDT can cause the variation of the species composition of copepods, and further affect the marine ecosystem.     

The effect of tributyltin upon steroid titres in the female dogwhelk, Nucella lapillus, and the development of imposex

Exposure of female Nucella lapillus to tributyltin (TBT) in seawater at a concentration of 40 ng Sn/litre led to accumulation of TBT in the tissues, and an increase in penis length compared to control animals. Investigation of the steroid concentrations by radioimmunoassay revealed a significant increase in testosterone after 28 days exposure to TBT. No significant effect was noted upon the amounts of progesterone or estradiol-17β. When testosterone was injected into female N. lapillus in the absence of TBT, penis length was found to increase significantly. These observation suggest that there may be an association between the change in testosterone titre in response to exposure to TBT and the development of imposex in the dogwhelk.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

Estrogenic responses of larval sunshine bass (Morone saxatilis x M. Chrysops) exposed to New York City sewage effluent

To determine the estrogenicity of effluents from sewage treatment plants (STPs) to larval fish, 2-day-old sunshine bass were exposed to effluents from three STPs serving New York City (NYC), varying in size and treatment level. Estrogenic response was evaluated by measuring vitellogenin (VTG) and estrogen receptor (ER) expression in cytosolic fractions of whole body homogenates. Concentrations of the presumptive endocrine disruptors in the effluents were also measured. VTG and ER levels in sewage-exposed fish were 3-5 times that observed in controls. Combined concentrations of estradiol and estrone ranged from 5 to 13 ng/l and nonylphenol-ethoxylate metabolites (NPEOs: 4-nonylphenol, and 1-, 2-, and 3-nonylphenol-ethoxylates) ranged from 180 to 470 microg/l in chlorinated effluent. Results indicate that both ER and VTG can be used as biomarkers for endocrine disruption in larval fish, and that 4-day exposure to sewage effluent is sufficient to elicit significant expression of these markers in sunshine bass larvae. The extremely higher concentrations of NPEOs found in effluent relative to hormones (approximately 40,000-fold) indicates that surfactant metabolites may be contributing significantly to the estrogenic effects observed.