Isolation and identification of ligands for the goldfish testis androgen receptor in chemical recovery condensates from a Canadian bleached kraft pulp and paper mill

This study builds on a series of investigations characterizing substances in kraft mill chemical recovery condensates that depress sex steroids in fish. Here, incubations of goldfish testis androgen receptors (AR) with condensate extracts were used to investigate the potential role of androgens in hormone depressions. Condensates contained variable levels of AR ligands, with the highest amounts in nonpolar extracts of filtered solids prior to solid phase extraction (SPE). High pressure liquid chromatography (HPLC) fractionation recovered the majority of activity in one fraction, with ligands detected in three additional fractions. Gas chromatography mass spectrometry analysis of the most active fraction confirmed the two most abundant components as the diterpenes manool and geranyl linalool. Manool exhibited a relative affinity for the AR that was 300 fold less than testosterone and accounted for 26% of total filtered solids activity. Geranyl linalool exhibited no affinity for the AR. Three additional diterpenoid families were tentatively identified as principal components of the three other androgenic HPLC fractions. Compared to condensates, final effluent had 3000 fold less androgenic activity, with <1% attributable to manool. Putative androgens previously associated with mill effluents (androstenedione and androstadienedione) and progesterone were not detected; however, additional condensate diterpenes suspected as androgens were identified in final effluent. This study is the first to confirm nonsteroidal cyclic diterpenes as androgenic at pulp mills. A major in-mill source of these substances was identified, and the role of androgens in mill effluents affecting fish reproduction was reinforced.     

Dairy wastewater, aquaculture, and spawning fish as sources of steroid hormones in the aquatic environment

A suite of androgens, estrogens, and progestins were measured in samples from dairy farms, aquaculture facilities, and surface waters with actively spawning fish using gas chromatography-tandem mass spectrometry (GC/MS/ MS) to assess the potential importance of these sources of steroid hormones to surface waters. In a dairywaste lagoon, the endogenous estrogens 17beta-estradiol and estrone and the androgens testosterone and androstenedione were detected at concentrations as high as 650 ng/L. Samples from nearby groundwater monitoring wells demonstrated removal of steroid hormones in the subsurface. Samples from nearby surface waters and tile drains likely impacted by animal wastes demonstrated the sporadic presence of the steroids 17beta-estradiol, estrone, testosterone, and medroxyprogesterone, usually at concentrations near or below 1 ng/L. The endogenous steroids estrone,testosterone, and androstenedione were detected in the raceways and effluents of three fish hatcheries at concentrations near 1 ng/L. Similar concentrations were detected in a river containing spawning adult Chinook salmon. These results indicate that dairy wastewater, aquaculture effluents, and even spawning fish can lead to detectable concentrations of steroid hormones in surface waters and that the concentrations of these compounds exhibit considerable temporal and spatial variation.     

Anaerobic biodegradation of sterols contained in kraft mill effluents

Bleached kraft mill requires large quantities of water and chemicals reactives and generates effluents containing active organic compounds. Specifically, hormonal changes in fish and their communities have been demonstrated, which could be attributed to endocrine-disrupting chemicals (EDCs) contained in kraft mill effluents. This chronic toxicity is attributable to extractive compounds, such as resin acids, sterols, and fatty acids. The goal of this work is to evaluate the methanogenic toxicity and anaerobic biodegradation of stigmasterol and beta-sitosterol. A continuously anaerobic filter (AF) was used for the anaerobic biodegradation of stigmasterol and beta-sitosterol. Three phases were evaluated. In phase I, an elementary chlorine-free bleached kraft mill effluent was fed to the reactor whereas in phases II and III, the effluent was supplemented with increasing stigmasterol and beta-sitosterol concentrations. The AF displays high performance in biological oxygen demand (BOD5) removal (up to 94%); however, only 50% of the chemical oxygen demand (COD) was removed. Simultaneously, the AF system shows a great ability to remove beta-sitosterols (77-100%) and stigmasterols (87-95%). No negative effect on the methanogenic activity inhibition was shown by beta-sitoesterols and stigmasterol. However, a mixture of beta-sitosterols and stigmasterols (ratio 1:1) caused a less than 10% reduction in methanogenic activity.     

Identification and functional characterisation of bioactive peptides in rice bran albumin hydrolysates

The potential heath‐benefitting bioactivities of rice bran albumin hydrolysates were investigated. The antioxidant and α‐glucosidase‐ and angiotensin‐converting enzyme (ACE)‐inhibitory activities of the crude hydrolysates and their fractions were determined. The fractions with the highest bioactivities were analysed by liquid chromatography–tandem mass spectrometry/mass spectrometry to identify the active peptide sequences. Hydrolysates produced by commercial proteases Alcalase and Protamax exhibited the highest α‐glucosidase‐ and ACE‐inhibitory activities, resulting in 43.1 ± 2.1% and 54.4 ± 5.1% inhibition of the enzymes, respectively. Inhibitory activities against both enzymes were highest in the MW<3‐kDa fractions that were eluted under alkaline conditions. A number of peptide sequences were identified in the fractions, which contained several sequences with reported α‐glucosidase‐ or ACE‐inhibitory activities. This is the first time that such activities are reported for rice bran albumin hydrolysate, and it demonstrated that the hydrolysates may be developed into nutraceuticals useful in managing diabetics and hypertension.     

Discrimination of aerial deposition sources of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran downwind from a pulp mill near Ketchikan, Alaska

Drinking water is supplied by individual roof-catchment systems for homes and businesses near a dissolving sulfite pulp mill (now closed) located just north of Ketchikan in southeast Alaska. This study was conducted to determine if polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/Fs) found in the sediments of the roof-catchment cisterns resulted from historical deposition of stack emissions from the pulp mill's multi-fuel power boilers. Fly ash from the power boilers had maximum total PCDD/F concentrations of 3.08 x 10(5)-3.10 x 10(6) ng/kg, which resulted from combustion of bleach plant wastewater sludge and saltwater-soaked wood waste. Cistern sediments had maximum total PCDD/F concentrations of 7.71 x 10(4) ng/kg. Potential sources of PCDDs/Fs in the cistern sediments were considered to be automobile exhaust, heating oil combustion, and private trash burning as well as pulp mill boiler emissions. Discriminant analysis was used to analyze differences between profiles of tetra through octa homologue classes of PCDDs/ Fs (defined as proportional contributions to total concentration) from different source terms. Homologue profiles of potential sources from Ketchikan included in this analysis were fly ash collected from the mill's power boilers and soils collected from background areas (areas with similar PCDD/F sources as the residences [e.g., auto exhaust and burn barrels] near the mill but beyond the zone of aerial deposition of emissions from the mill). Profiles for emissions from automobile exhaust, fertilizers, oil heating, residential trash burning, and residential wood heating were also included in the source "training" data set (for the discriminant analysis) using data from published literature. The classification rules developed from the discriminant analysis were applied to the following test media sampled at Ketchikan: roof-catchment cistern sediments and soils collected from areas in the vicinity of the mill's power boilers (i.e., nearby residential or commercial [developed] areas, on the mill property, and nearby forestlands). The homologue profiles of cistern sediment and nearby developed area soil samples were similar to background soils, whereas the profiles for the forestland soil samples (influenced by emissions from the mill but not other anthropogenic sources) closely matched the fly ash pattern. The homologue profiles of the emission sources from published data were more similar to one another than either background soils or fly ash. Soil samples from the mill property were classified as members of all source groups. On the basis of these analyses, the composition of PCDDs/Fs detected in the cistern sediments is typical of Ketchikan background conditions and not reflective of mill emissions.     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of egg protein hydrolysates produced with gastrointestinal and nongastrointestinal enzymes

Egg is a well-known rich source of bioactive peptides. In this study, egg protein (egg white and egg yolk proteins) hydrolysates were produced with gastrointestinal enzymes (pepsin and pancreatin) or nongastrointestinal enzymes (thermolysin and alcalase), and fractionated by ultrafiltration and cation exchange chromatography. Angiotensin-I converting enzyme (ACE) inhibitory and antioxidant activities, amino acid composition and molecular weight distribution were studied, and the physicochemical properties were related with the bioactivities. Our results showed that egg protein hydrolysates produced with non-GI enzymes (thermolysin and alcalase) showed significantly higher ACE inhibitory activity, whereas similar or even lower antioxidative activities, than those of hydrolysates produced with GI enzymes. ACE-inhibitory activity significantly correlated with the amino acid composition, especially the proportion of positively charged amino acid, whereas antioxidant activities correlated with the proportion of low molecular weight peptides under 500 Da. Understanding the relationship between the bioactivities and physicochemical properties of the hydrolysates/fractions is important to facilitate the development technologies for preparing fractions with improved bioactivities.     

Comparison of Effect of Gear Juicer and Colloid Mill on Microstructure,Polyphenols Profile,and Bioactivities of Mulberry (<Emphasis Type="Italic">Morus indica L</Emphasis>.)

Mulberry (Morus indica L.) fruits are rich sources of polyphenols with multiple health benefits. The gear juicer and colloid mill were compared in mulberry juice production and further marc water extraction based on polyphenol yield, phytochemical profiles, bioactivities, as well as microstructure of residues. Higher content of phenolic compounds, stronger antioxidative, and α-Glucosidase inhibition activities were obtained in colloid mill-processed juice. The extraction kinetics of marc was investigated under three conditions (stirring, ultrasound, and combined stirring and ultrasound) at 25 °C for 2.5 h. Based on total polyphenol and monomeric anthocyanin yield, the optimum extraction condition for gear juicer marc was combination of ultrasound with stirring for half an hour. While ultrasound for 1 h was optimized for water extraction of colloid mill marc. Using the optimized extraction condition, total polyphenols and monomeric anthocyanin obtained from colloid mill-processed samples, including juice and marc extract, were approximately 46 and 68 % higher than those in gear juicer-processed samples. Compared to gear juicer, colloid mill processing generated smaller particle size and more disrupted microstructure, which contributed to higher content of phenolic compounds in colloid mill juice. Moreover, changes in the microstructure of mulberry marc were intensified by extraction conditions, which improved polyphenol extraction efficiency, especially for colloid mill marc. These results provided valuable information for comparing gear juicer and colloid mill in mulberry as well as other berries for juice production and marc water extraction, and emphasized the great potential of colloid mill in berry processing.     

Assessment of the reproductive-endocrine disrupting potential of chlorine dioxide oxidation products of plant sterols

This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta-sitosterol and stigmasterol. Oxidation products of the plant sterol beta-sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms.     

Global gene expression profiling of androgen disruption in Qurt strain medaka

Androgen disrupting chemicals (ADCs) are endocrine disrupting chemicals (EDCs) that mimic or antagonize the effect of physiological androgens. Microarray-based detection of altered gene expression can be used as a biomarker of EDC exposure. Therefore, the purpose of this study was to identify and compare gene expression profiles of the androgen 11-ketotestosterone (11-KT), the antiandrogen flutamide (FLU), and the antiandrogenic fungicide vinclozolin (VIN), on Qurt medaka (Oryzias latipes). Biologically effective concentrations for 11-KT (100 microg/L), VIN (100 microg/L), and FLU (1000 microg/L) determined in range-finding studies were used for exposures. The oligonucleotide microarray included 9379 probes for EDC-affected genes, medaka cDNAs, sequences from the medaka genome project, and the UniGene database. We found that treatment with FLU, VIN, and 11-KT caused significant (false discovery rate = 0.01) differential expression of at least 87, 82, and 578 genes, respectively. Two sets of responsive genes are associated to vertebrate sex differentiation and growth, and 50 genes were useful in discriminating between ADC classes. The discriminating capacity was confirmed by a remarkable similarity of the antiandrogenic expression profiles of VIN and FLU, which were distinct from the androgenic profile of 11-KT. Gene expression profiles characterized in this study allow for reliable screening of ADC activity.     

Bioactivities of peptide fractions derived from proteolytic enzyme-injected Hanwoo longissimus muscle in a model system

In this study, crude peptide fractions from Hanwoo loins were released by injecting with proteolytic enzymes [no enzymes (control); protease type XIII (E1); thermolysin (E2); and combination of E1 and E2 (E3)] and their bioactivities were determined. The peptides derived from E2-injected Hanwoo loin exhibited the highest angiotensin I–converting enzyme (ACE) inhibitory activity and vitamin C equivalents antioxidant capacity among the treatments. The released peptide by treatment of E2 and E3 had similar (P > 0.05) inhibitory activity in HT29 cancer cell viability compared with luteolin as a positive control and non-cytotoxic effect on normal cell (3T3-L1). Therefore, the released peptide fraction from thermolysin (E2)-injected Hanwoo beef might contain potent bioactive peptides with ACE inhibitory and antioxidative activity and inhibition effect on certain cancer cell viability.     

Extraction,optimisation and characterisation of phenolics from Thymus vulgaris L.: phenolic content and profiles in relation to antioxidant,antidiabetic and antihypertensive properties

The objectives of this study were to examine varying extraction conditions of Thymus vulgaris L. as related to phenolic content and profiles of the extracts and their antioxidant, antihypertensive and antidiabetic properties. Phenolics were extracted under various conditions pertaining to free and bound phenolics, solvent type and combination of extraction time and temperature, and these extracts were evaluated in terms of their antioxidant activities and inhibitory activities of angiotensin‐converting enzyme (ACE), α‐glucosidase and α‐amylase. The acetone–water solvent mixture (1:1; v/v) produced the extract with the greatest phenolic content, antioxidant activity and inhibitory activities of ACE and α‐glucosidase. The optimal extraction temperature for maximum phenolic content and antioxidant activity associated with methanol extraction was 60 °C, whereas a lower temperature at 40 °C was required to maximise inhibitory activities for ACE, α‐glucosidase and α‐amylase. An inverse relationship was seen between antioxidant and glucosidase inhibitory activities vs. the ACE and α‐amylase inhibitory activities, which suggests the need for extractions to be directed to specific bioactivities of thyme extracts. Generally, the results indicate major differences in phenolic profiles among the tested extraction conditions with thymol as the predominant phenolic seen in most extractions, while gallic acid, rosmarinic acid or diosmin also predominated in other extracts. Extracts with the same predominant phenolic compound and similar phenolic content showed major disparities in their ACE, glucosidase and α‐amylase inhibitory activities, indicating that the major phenolic profiles of thyme extracts may not be necessarily related to the degree of inhibition of ACE, glucosidase and α‐amylase enzymes.     

Comparison of ACE inhibitory and DPPH radical scavenging activities of fish muscle hydrolysates

To utilise Atlantic salmon, Coho salmon, Alaska pollack, and southern blue whiting as components of neutraceutical food and to clarify the potential physiological function of those fishes, their muscles were hydrolysed with pepsin, pancreatin or thermolysin. Methanolic extracts of fish muscle and their hydrolysates were prepared for analysis of angiotensin converting enzyme (ACE) inhibitory and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. Pepsin hydrolysates of all the fish samples did not increase degree of hydrolysis, extractive nitrogen content and bioactivities. Pancreatin and thermolysin improved the ACE inhibitory activity, and the activity was expressed following the peak of size exclusion chromatography (SEC). The DPPH radical scavenging activity was increased following pancreatin and thermolysin hydrolysis, but this activity was not related to the SEC result. Except for the lower DPPH radical scavenging activity of Alaska pollack pancreatin hydrolysate than those of the others, there was no significant difference in the ACE inhibitory and DPPH radical scavenging activities by fish species.     

A HPLC-UV method for the determination of angiotensin I-converting enzyme (ACE) inhibitory activity

To determine the angiotensin-converting enzyme (ACE) inhibitory activity of a fish hydrolysate, different methods were tested. Finally, a sensitive, extraction-free HPLC method using N-(3-[2-furylacryloyl)-Phe-Gly-Gly (FAPGG) as substrate was preferred. This method relies on the UV-titration of the peptide 2-furylacryloyl-l-Phe (FAP) resulting from the hydrolysis of the FAPGG after a chromatographic separation on a reverse phase column. The experimental conditions (enzyme/substrate ratio, incubation time, NaCl concentration) were optimised for linearity, sensitivity and precision. The assay was adequate for the study of ACE inhibition by Captopril, used as reference, and several peptides. Captopril and the fish hydrolysate had IC₅₀ values, respectively of 0.19 ng and 43 μg with standard deviations of 0.09 ng and 5 μg. Afterwards, the determination of the Hill coefficient sustained the hypothesis that active peptides present in the fish hydrolysate were low-molecular weight molecules. This result was confirmed by the activity measurement of the fish hydrolysate fractions obtained by gel filtration.     

Assessment of endogenous androgen levels in meat,liver and testis of Iranian native cross-breed male sheep and bull by gas chromatography-mass spectrometry

Androgenic steroids always exist in different animal tissues at trace level, with significant numbers of interfering compounds, which makes their determination difficult. To solve some of the problems in quantification of the natural steroids in those tissues, a new GC-MS method was developed in this study. By using a surrogate analyte approach, which was developed in the authors’ previous studies, and extensive sample preparation procedure, which successfully eliminates many of the interfering compounds and resulting in a cleaner extract, accuracy, precision, sensitivity and selectivity of the method for the determination of steroids in complex matrices such as meat, liver and testis were improved. By aid of this method, the levels of androgens in different tissues of Iranian native cross-breed bulls and male sheep were determined. According to the results obtained in the present study, although the androgenic profile (contents and ratios of precursors and metabolites to the main hormones) is similar between the same tissues of both animals, the total androgenic content of each tissue is higher in the bull than the same tissue in male sheep. In addition, in both animals higher amount of androgens were found in liver in comparison with meat and testis.     

Effect of different pasteurization conditions on bioactivities of Lupinus albus protein isolates

Lupin protein isolate was extracted following the procedure in European Patent (EP 1024 706 B1) in order to use lupin protein for food and pharmaceutical applications. The acid insoluble/neutral pH soluble protein isolate was pasteurized at 65-125 °C for 10-1000 s. The objective of this study is finding out reasonable pasteurization condition for food use, or for good bioactivities like radical scavenging, angiotensin converting enzyme inhibition, and bile acid binding activity. Pasteurization at 65 °C for 10 s did not reduce the microbial count of the protein sufficiently for use in foods. The chemical composition of lupin protein isolates had no change by various pasteurization. The angiotensin converting enzyme inhibition decreased and the DPPH radical scavenging capacity increased after high temperature treatment at 125 °C. The sodium cholate binding capacity was not affected by tested conditions. Pasteurization at higher temperature is useful for producing selective bioactive fractions with suitable microbiological properties.     

Gene expression profiling for understanding chemical causation of biological effects for complex mixtures: a case study on estrogens

Gene expression profiling offers considerable potential for identifying chemical causation of effects induced in exposures to complex mixtures, and for understanding the mechanistic basis for their phenotypic effects. We characterized gene expression responses in livers and gonads of fathead minnow (Pimephales promelas) exposed (for 14-21 days) to estrogenic wastewater treatment works final effluents with varying potencies and assessed the extent to which these expression profiles mapped with those induced by individual steroid estrogens present in the effluents (17beta-estradiol and 17alpha-ethinylestradiol) and, thus, were diagnostic of estrogen exposure. For these studies, we adopted a targeted approach (via real-time PCR) with a suite of 12 genes in liver and 21 genes in gonad known to play key roles in reproduction, growth and development (processes controlled by estrogens) and responses were compared with effects on phenotypic end points indicative of feminization. Gene responses to effluent were induced predominantly in a linear (monotonic) concentration-dependent manner but were complex with many genes responding differently between tissue types and sexes. The gene expression profiles for the estrogenic effluents and the individual steroid estrogens had many common features. There were marked differences in the profiles between the two effluents, however, that were not explained by differences in their estrogenic potencies, suggesting that these may have arisen as a consequence of differences in the contents of other chemicals, which may act directly or indirectly with the estrogen-response pathway to alter estrogen-induced gene expression. These data demonstrate that the patterns of gene expression induced by estrogenic effluents, although complex, can be diagnostic for some of the estrogens they contain and provide insights into the mechanistic basis for the phenotypic effects seen.     

生物精炼在造纸工业中的应用模式和发展趋势

对生物精炼技术的概念和木材生物精炼工厂（IFBR）的路线进行了阐述。对传统硫酸盐法制浆造纸工厂如何转化为综合林业生物精炼工厂的原理进行了分析,总结出三种转化类型。详细介绍了近中性预抽提/制浆模式、酸性预水解/制浆模式和碱预处理纤维素糖化发酵生产乙醇模式。保留制浆造纸生产的综合林业生物精炼工厂将是近期主要的发展模式,可为传统造纸工厂带来额外的经济收益。在传统制浆之前进行预抽提,分离出的抽提液可以用来生产乙醇和乙酸及其它化学品,抽提后的木片进行制浆和漂白,对纸浆强度和光学指标没有不良影响,新增用于改造的投资回报率达到7.1%～13%。根据我国造纸工业特点,木材造纸工厂转化为IFBR工厂也将走保留制浆的发展模式;草类原料造纸工厂转化为生物精炼工厂具有一定的优势和可行性。     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.     

Accounting for differences in estrogenic responses in rainbow trout (Oncorhynchus mykiss: Salmonidae) and roach (Rutilus rutilus: Cyprinidae) exposed to effluents from wastewater treatment works

Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW A and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.