Response of periphytic biofilm in water to estrone exposure: Phenomenon and mechanism.

The responses of pure strains to contaminant (i.e., estrone, E1) exposure have been widely studied. However, few studies about the responses of multispecies microbial aggregates (e.g., periphytic biofilm) to E1 exposure are available. In this study, the changes in physiological activity and community composition of periphytic biofilms before and after E1 exposure were investigated. The results showed that periphytic biofilms exhibited high adaptability to E1 exposure at a concentration of 0.5 mg L-1 based on physiological results. The increase in productivity of extracellular polymeric substances (EPS) after exposure to E1 was the main factor preventing association between E1 and microbial cells. The increase in the activity of superoxide dismutase (SOD) and ATP enzyme activity and the change in the co-occurrence pattern of microbial communities (increasing the relative abundance of Xanthomonadaceae and Cryomorphacea) also protected biofilms from E1 exposure. However, exposure to a high concentration of E1 (>10 mg L-1) significantly decreased EPS productivity and metabolic activity due to the excessive accumulation of reactive oxygen species. In addition, the abundance of some sensitive species, such as Pseudanabaenaceae, decreased sharply at this concentration. Overall, this study highlighted the feasibility of periphytic biofilms to adapt to E1 exposure at low concentrations in aquatic environments.

Opposite estrogen effects of estrone and 2-hydroxyestrone on MCF-7 sensitivity to the cytotoxic action of cell growth, oxidative stress and inflammation activity.

There are many kinds of estrogens, and endogenous estrogens produce a variety of estrogen metabolites with similar structure but with different physiological effects after metabolism in vivo. Studies have shown that estrone (E1) widely occurs in the environment and animal-derived food. Because of its estrogen effect, E1 can have adverse effects on the human body as an endocrine disruptor. In this study, we found that E1 and 2-hydroxyestrone (2-OH-E1), the hydroxylation metabolite of estrogen, have opposite proliferative effects on breast cancer cells (MCF-7) through cell proliferation experiments and comparison of their effects by molecular docking and detection of ROS, Ca2+, and cell pathway proteins. The effects of 2-methoxyestrone (2-MeO-E1) and 16α-hydroxyestrone (16α-OH-E1) on the biochemical and protein levels of MCF-7 were further studied to compare the effects of metabolic sites and modes on estrogen effects. Hydroxylation of E1 at the C2 site weakened the estrogen effect, down-regulated the expression of the mammalian target of rapamycin (mTOR) and protein kinase B (Akt) pathway proteins, inhibited the proliferation of cancer cells, and enhanced anti-oxidative stress and anti-inflammation. Methoxylation at the C2 position also inhibited the expression of inflammatory and oxidative stress pathway proteins but did not greatly affect the estrogen effects. However, hydroxylation on C16 had no significant effect on the biological effects of estrogen. Therefore, the structural changes of estrogen on C2 are important reasons for the different physiological effects of estrogen and its metabolites. Thus, by regulating the gene Cytochrome P450 1B1(CYP1B1), which affects the hydroxylation metabolism of estrogen, and promoting the hydroxylation of estrone at the C2 position, the estrogen effect of estrone can be effectively reduced, thus reducing the harm its poses in food and the environment.

The 3,4-Quinones of Estrone and Estradiol Are the Initiators of Cancer whereas Resveratrol and N-acetylcysteine Are the Preventers.

This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the form of catechol estrogen-3,4-quinones, play a central role in this pathway of cancer initiation. The catechol estrogen-3,4-quinones react with specific purine bases in DNA to form depurinating estrogen-DNA adducts that generate apurinic sites. The apurinic sites can then lead to cancer-causing mutations. The process of cancer initiation has been demonstrated using results from test tube reactions, cultured mammalian cells, and human subjects. Increased amounts of estrogen-DNA adducts are found not only in people with several different types of cancer but also in women at high risk for breast cancer, indicating that the formation of adducts is on the pathway to cancer initiation. Two compounds, resveratrol, and N-acetylcysteine, are particularly good at preventing the formation of estrogen-DNA adducts in humans and are, thus, potential cancer-prevention compounds.

Evaluation of the oestrogenic potential of oestrone and bisphenol-A on the reproduction of Astyanax bimaculatus males after subacute exposure.

In the last decades, oestrogenic compounds have often been reported in environmentally relevant concentrations in aquatic environments around the world. Most laboratory studies of oestrogens try to understand the effects of a single contaminant, but in natural environments, the effects may be quite different due to interactions with other compounds. The present study aimed to compare the action of oestrone (E1) and bisphenol-A (BPA), acting singularly and in combination, on the spermatogenesis of Astyanax bimaculatus. After exposure to 100 ng/L of E1, BPA and a mixture of the two for 15 days, our results showed that E1 and the E1 + BPA mixture significantly altered the number of spermatogenic cells. BPA presented high cytotoxicity when compared to other treatments. Analysis of the two oestrogenic compounds suggests that the E1 + BPA mixture has no additive or synergistic effects. Together, the results of the present study indicate that endocrine-disrupting chemicals (EDCs) analysed alone may behave differently than when administered with other substances.

Temperature-Dependent Biomarkers of Estrogenic Exposure in a Piscivore Freshwater Fish.

The biological effects of endocrine-active compounds and increasing water temperatures as a result of climate change have been studied extensively and independently, but there is a dearth of research to examine the combined effect of these factors on exposed organisms. Recent data suggest that estrogenic exposure and rising ambient temperatures independently impact predator-prey relationships. However, establishing these connections in natural settings is complex. These obstacles can be circumvented if biomarkers of estrogenic exposure in resident fish can predict changes in predator-prey relationships. To test the effects of estrone and temperature, the piscivore bluegill sunfish (Lepomis macrochirus) was exposed for 30 days to estrone at concentrations (90 ± 17.6 ng/L [mean ± standard deviation] and 414 ± 146 ng/L) previously shown to reduce prey-capture success. Exposures were conducted at four temperatures (15 °C, 18 °C, 21 °C, 24 °C) to simulate breeding season ambient temperatures across the natural range of this species. A suite of morphological and physiological biomarkers previously linked to estrogenic exposures were examined. Biomarkers of estrone exposure were more commonly and severely impacted in male fish than in female fish. Notably, the gonadosomatic index was lower and gonads were less mature in exposed males. Additionally, temperature modulated the effects of estrone similarly in males and females with fish exposed at higher temperatures typically exhibiting a decreased morphological index. This study provides evidence that alterations in hepatic function and gonadal function may cause shifts in metabolism and energy allocation that may lead to declining prey capture performance.

Synthesis, and anti-proliferative, Pim-1 kinase inhibitors and molecular docking of thiophenes derived from estrone.

Heterocyclization of steroids were reported to give biologically active products where ring D modification occured. Estrone (1) was used as a template to develop new heterocyclic compounds. Ring D modification of 1 through its reaction with cyanoacetylhydrazine and elemental sulfur gave the thiophene derivative 3. The latter compound reacted with acetophenone derivatives 4a-c to give the hydrazide-hydrazone derivatives 5a-c, respectively. In addition, compound 3 formed thiazole derivatives through its first reaction with phenylisothiocyanate to give the thiourea derivative 9 followed by the reaction of the later with α-halocarbonyl compounds. In the present work a series of novel estrone derivatives were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase, and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). The most promising compounds 5b, 5c, 11a, 13c, 15b, 15c, 15d, 17a and 17b were further investigated against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. Compounds 5b, 15d, 17a and 17b were selected to examine their Pim-1 kinase inhibition activity where compounds 15d and 17b showed high activities. Molecular docking of some of the most potent compounds was demonstrated.

Estrogenic activity, selected plasticizers and potential health risks associated with bottled water in South Africa.

Potential endocrine disrupting chemicals (EDCs) are present in bottled water from various countries. In South Africa (SA), increased bottled water consumption and concomitant increases in plastic packaging create important consequences for public health. This study aimed to screen SA bottled water for estrogenic activity, selected target chemicals and assessing potential health risks. Ten bottled water brands were exposed to 20 °C and 40 °C over 10 days. Estrogenic activity was assessed using the recombinant yeast estrogen screen (YES) and the T47D-KBluc reporter gene assay. Solid phase extracts of samples were analyzed for bis(2-ethylhexyl) adipate (DEHA), selected phthalates, bisphenol-A (BPA), 4-nonylphenol (4-NP), 17ß-estradiol (E2), estrone (E1), and ethynylestradiol (EE2) using gas chromatography-mass spectrophotometry. Using a scenario-based health risk assessment, human health risks associated with bottled water consumption were evaluated. Estrogenic activity was detected at 20 °C (n = 2) and at 40 °C (n = 8). Estradiol equivalent (EEq) values ranged from 0.001 to 0.003 ng/L. BPA concentrations ranged from 0.9 ng/L to 10.06 ng/L. Although EEqs and BPA concentrations were higher in bottled water stored at 40 °C compared to 20 °C, samples posed an acceptable risk for a lifetime of exposure. Irrespective of temperature, bottled water from SA contained chemicals with acceptable health risks.

Divergent responsiveness of two isoforms of the estrogen receptor to mixtures of contaminants of emerging concern in four vertebrates.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17ß, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erß) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17ß. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species.

Fish multigeneration test with preliminary short-term reproduction assay for estrone using Japanese medaka (Oryzias latipes).

The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste.Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l(­1) of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long-term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l(­1). No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l(­1) estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l(­1), suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis­ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis-ovais regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption.

Effects of single and mixed estrogens on single and combined cultures of D. subspicatus and P. subcapitata.

This paper investigates the effect of estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) individually and mixed at equal proportions (1:1:1) on Desmodesmus subspicatus and Pseudokirchneriella subcapitata in single and combined cultures (S+) at different exposure times based on algal growth (in vivo chlorophyll fluorescence and cell counting) and coenobium formation. EE2 and E2 were more toxic to individual and combined (S+) cultures than was E1. The frequency of coenobium formation by D. subspicatus increased significantly for all estrogens and all concentrations. After 96 h, D. subspicatus prevailed in S+. The results of the exposure to E+ suggested a less-than-additive effect on D. subspicatus and S+ and additive effect on P. subcapitata. Toxic effects occurred for both species exposed to E+ with individual estrogen concentrations below the NOEC of each species. Assays must include changes in response due to the exposure of more than one species to more than one estrogen.

Exposure to estrone disrupts the endocrine system of western mosquitofish (Gambusia affinis).

Estrone (E1) is one of the predominant natural estrogens detected in aquatic environments, yet little is known about its effects on the endocrine system in fish. In this study, the sex ratio, secondary sexual characteristics, gonadal histology, and transcriptional levels of genes closely related to sex differentiation and hypothalamic-pituitary-gonadal-liver (HPGL) axis were assessed in western mosquitofish (Gambusia affinis) after a full life-cycle exposure to E1 (0, 25.4, 143, 740, and 4300 ng/L) for 119 days. The results showed that exposure to 4300 ng/L of E1 resulted in 100% female and inhibited the growth of females. Exposure to environmentally relevant concentrations of E1 (143 and 740 ng/L) led to obvious feminization of skeletons and anal fins in males. Exposure to 740 and 4300 ng/L of E1 increased the proportion of mature spermatocytes in females, and exposure to 143 and 740 ng/L decreased the proportion of mature spermatocytes in males. Moreover, the transcripts of genes related to sex differentiation and HPGL axis were changed in the E1-exposed adult fish and embryos inside females. This study has provided valuable data on the endocrine disruption effects of E1 at environmentally relevant concentrations in G. affinis.

Bisphenol A and estrone-induced developmental effects in early chick embryos.

Endocrine disruptors, especially estrogenic substances, are thought to affect the reproduction and development of animals, including humans. We therefore assessed whether bisphenol A (BPA) or estrone (E1) had any adverse effects on chick embryogenesis. Fertilized eggs of white Leghorns were obtained within 24 h after laying. Embryos were administered 0.1, 1.0, and 10.0 mM BPA, and administered 10, 100 nM, and 1 µM E1, and incubated for 48 h at 37 ± 0.5°C and >80% relative humidity with one rotation per hour. The embryos were excised, fixed in 70% ethanol and viewed under a stereomicroscope. Their morphological abnormalities and numbers of somites were recorded. There were no significant difference in the average number of somites in embryos administered BPA and controls. Abnormal embryogenesis, however, showed dose-related increases caused by BPA and E1.

Occurrence and removal efficiencies of eight EDCs and estrogenicity in a STP.

The occurrence and removal of eight endocrine disrupting compounds (EDCs), including estrone (E(1)), 17ß-estradiol (E(2)), estriol (E(3)), 17α-ethinylestradiol (EE(2)), diethylstilbestrol (DES), bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP), and their estrogenicities were investigated in a sewage treatment plant in Harbin city, China. The EDCs were extracted from wastewater samples by solid phase extraction (SPE) method and analyzed with gas chromatography coupled with mass spectrometry (GC-MS). The average concentrations in the influents and effluents ranged from 6.3 (EE(2)) to 1725.8 ng L(-1) (NP) and from

The phenotypic and transcriptomic effects of developmental exposure to nanomolar levels of estrone and bisphenol A in zebrafish.

Estrone and BPA are two endocrine disrupting chemicals (EDCs) that are predicted to be less potent than estrogens such as 17ß-estradiol and 17α-ethinylestradiol. Human exposure concentrations to estrone and BPA can be as low as nanomolar levels. However, very few toxicological studies have focused on the nanomolar-dose effects. Low level of EDCs can potentially cause non-monotonic responses. In addition, exposures at different developmental stages can lead to different health outcomes. To identify the nanomolar-dose effects of estrone and BPA, we used zebrafish modeling to study the phenotypic and transcriptomic responses after extended duration exposure from 0 to 5 days post-fertilization (dpf) and short-term exposure at days 4-5 post fertilization. We found that non-monotonic transcriptomic responses occurred after extended duration exposures at 1 nM of estrone or BPA. At this level, estrone also caused hypoactivity locomotive behavior in zebrafish. After both extended duration and short-term exposures, BPA led to more apparent phenotypic responses, i.e. skeletal abnormalities and locomotion changes, and more significant transcriptomic responses than estrone exposure. After short-term exposure, BPA at concentrations equal or above 100 nM affected locomotive behavior and changed the expression of both estrogenic and non-estrogenic genes that are linked to neurological diseases. These data provide gaps of mechanisms between neurological genes expression and associated phenotypic response due to estrone or BPA exposures. This study also provides insights for assessing the acceptable concentration of BPA and estrone in aquatic environments.

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 1. Dissolved oxygen and sodium chloride.

Laboratory exposures indicate that estrogens and their mimics can cause endocrine disruption in male fishes, yet while studies of resident fish populations in estrogen-polluted waters support these findings, biomarker expression associated with field versus laboratory exposure to estrogenic endocrine disruptors (EDs) often differ dramatically. Two of the environmental parameters often found to vary in dynamic aquatic ecosystems were chosen (dissolved oxygen [DO] and sodium chloride concentrations) to assess their potential impact on ED exposure. In separate experiments, male fathead minnows (Pimephales promelas) were exposed to estrone (E1) a natural ED, under either two concentrations of DO, or two concentrations of sodium chloride, in a laboratory flow-through system. Morphological and hematological parameters were assessed. While vitellogenin concentrations were elevated with exposure to estrone (29 to 390ng/L), the effect on other indices were variable. Estrone exposure altered SSC, blood glucose, hematocrit, and hepatic and gonado-somatic index in 1 of 4 experiments, while it decreased body condition factor in 3 of 4 experiments. At the concentrations tested, no main effect differences (P<0.05) were found associated with DO or sodium chloride treatments, except in one experiment low DO resulted in a decrease in secondary sex characteristic score (SSC). The combination of DO or sodium chloride and E1 altered blood glucose in one experiment each. These results indicate the variability of fathead minnow response to estrone, even within the confines of controlled laboratory conditions.

Modeling the exposure of wild fish to endocrine active chemicals: Potential linkages of total estrogenicity to field-observed intersex.

Decades of studies on endocrine disruption have suggested the need to manage the release of key estrogens from municipal wastewater treatment plants (WWTP). However, the proposed thresholds are below the detection limits of most routine chemical analysis, thereby restricting the ability of watershed managers to assess the environmental exposure appropriately. In this study, we demonstrated the utility of a mechanistic model to address the data gaps on estrogen exposure. Concentrations of the prominent estrogenic contaminants in wastewaters (estrone, estradiol, and ethinylestradiol) were simulated in the Grand River in southern Ontario (Canada) for nine years, including a period when major WWTP upgrades occurred. The predicted concentrations expressed as total estrogenicity (E2 equivalent concentrations) were contrasted to a key estrogenic response (i.e., intersex) in rainbow darter (Etheostoma caeruleum), a wild sentinel fish species. A predicted total estrogenicity in the river of ≥10 ng/L E2 equivalents was associated with high intersex incidence and severity, whereas concentrations <0.1 ng/L E2 equivalents were associated with minimal intersex expression. Exposure to a predicted river concentration of 0.4 ng/L E2 equivalents, the environmental quality standard (EQS) proposed by the European Union for estradiol, was associated with 34% (95% CI:30-38) intersex incidence and a very low severity score of 0.6 (95% CI:0.5-0.7). This exposure is not predicted to cause adverse effects in rainbow darter. The analyses completed in this study were only based on the predicted presence of three major estrogens (E1, E2, EE2), so caution must be exercised when interpreting the results. Nevertheless, this study illustrates the use of models for exposure assessment, especially when measured data are not available.

Occurrence and ecological risk assessment of 22 emerging contaminants in the Jilin Songhua River (Northeast China).

Rivers may receive pharmaceuticals, personal care products, and environment estrogens, which are emerging concerns, from various sources. Understanding the fate of these emerging contaminants (ECs) from the sources to their receiving river is important for assessing their ecosystem risk. Here, the occurrence, seasonal variation, spatial distribution, and ecological risk of 22 ECs in water and sediments from the Jilin Songhua River, as well as in the effluents from the riverside Jilin wastewater treatment plant (WWTP) were investigated. Results indicated that estriol with the highest median concentration of 21.5 ng L-1 in the river water and with the highest median concentration of 481.5 ng g-1 in the sediments, and methylparaben with the highest concentration of 29.6 ± 2.9 ng L-1 in the WWTP effluents were the predominant contaminants. The total concentration of ECs in the river water in the dry season was about 1.5 times higher than that in the wet season. The concentrations of these ECs close to the contaminated tributary and the WWTP were relatively high. Risk assessment showed that the maximum risk quotient value of estrone was 1.07 in the river water and estriol was 2.10 in the effluents. In addition, erythromycin posed generally medium risk in the river water and WWTP effluents. It should be paid attention to the prior control of the three contaminants in the river region.

Associations between altered vitellogenin concentrations and adverse health effects in fathead minnow (Pimephales promelas).

Mechanism specific biomarkers are used in ecotoxicology to identify classes of chemicals and to inform on their presence in the environment, but their use in signalling for adverse effects has been limited by a poor understanding of their associated links with health. In this study an experimental analysis was undertaken to investigate how induction or suppression of an estrogen-dependent biomarker, vitellogenin (VTG), related to health effects in fathead minnow (Pimephales promelas, FHM). Exposure to an oestrogen agonist, estradiol (29 and 60 ng/L), resulted in rapid induction of VTG (elevated plasma concentrations within 2 days of exposure) in male FHM that was subsequently slow to clear from the plasma (concentrations remained elevated 70 days after cessation of exposure). The induction of VTG to concentrations of 0.5 mg/mL, however, and its continued presence in the plasma were not associated with any overt adverse health effects to the males. In contrast, induction of higher concentrations of VTG (>1 mg/mL) in reproductively active FHM exposed to estrone (307 and 781 ng/L), were associated with impacts on male survival (>33% male mortality) and an inhibitory effect on egg production in females (>51% decrease in egg number). Exposure of reproductively active FHM to a chemical that disrupts estrogen biosynthesis (an aromatase inhibitor; fenarimol 497 microg/L) also reduced reproductive success (40% decrease in egg number), and this was associated with a reduction in plasma VTG concentrations in females (36% decrease). These findings show that high level induction or suppression (in females) of plasma VTG are associated with alterations in health status and reproductive fitness. VTG, therefore, has the potential to act as a health measure as well as a biomarker for exposure, for chemicals that alter the oestrogen signalling pathway.

Effects of estrone on full life cycle of Java medaka (Oryzias javanicus), a new marine test fish.

Estrone is a natural estrogen detected in sewage treatment works effluents and in estuarine waters. However, there is little information on the effects of estrone on marine fish. This study investigated the effects of estrone on reproduction of the estuarine fish, Java medaka (Oryzias javanicus). Java medaka were exposed to concentrations of 39, 198, 484, 1,188, and 3,701 ng/L of estrone from embryonic stages up to adult stages for 239 d after hatching. The fertility and egg numbers of Java medaka exposed to 1,188 and 3,701 ng/L were significantly lower than that of control. The hepatic vitellogenin concentrations in male Java medaka exposed to estrone greater than 484 ng/L were significantly higher than that of control. Oocytes in testis (testis-ova) were not detected in the males in any of the exposure groups. The lowest-observed-effect concentration and no-observed-effect concentration for Java medaka were 484 and 198 ng/L of estrone. These results suggest that in relatively low estrone concentrations, 39 and 198 ng/L, Java medaka will not be affected by exposure to estrone.

Biomonitoring of estrogenic exposure and identification of responsible compounds in bream from Dutch surface waters.

The exposure to and effects of estrogenic compounds in male breams from Dutch freshwater locations were investigated. Ovotestis was observed infrequently (maximum frequency 16%). However, plasma vitellogenin (VTG) concentration was elevated highly at some locations. Estrogenic activities in male bream plasma, liver, and in gastrointestinal content were measured in the estrogen-responsive chemical-activated luciferase gene expression (ER-CALUX) assay. Plasma concentrations of vitellogenin correlated very well with the estrogenic activities in gastrointestinal content. The ER-CALUX activity in gastrointestinal content thus could provide a biomarker for recent exposure to estrogenic compounds, and the gastrointestinal content was chosen as investigative matrix for the toxicity identification and evaluation ([TIE]; bioassay-directed fractionation) of estrogenic compounds in bream. The approach consisted of a reversed-phase high-performance liquid chromatography fractionation of gastrointestinal content extract, directed by ER-CALUX and followed by gas chromatography analysis. The estrogenic hormones 17beta-estradiol and its metabolite estrone were identified as major contributors to the activity at all locations (except the reference location), independent of the presence or absence of a known source of estrogenic activity, such as a sewage treatment plant. Chemical screening showed the presence of other pollutants, such as a lower chlorinated dioxin and the disinfectants clorophene and triclosan. However, these compounds did not have high estrogenic potencies and their concentrations were not high enough to contribute significantly to the observed estrogenic activity.