Evaluation of the Ishikawa cell line bioassay for the detection of estrogenic substances from sediment extracts

This study examines the application of Ishikawa human endometrial adenocarcinoma cells to measure the estrogenic activity of fractionated extracts of sediments from Tokyo Bay, Japan. Estrogen stimulates alkaline phosphatase activity in this cell line. The results of these assays were compared with those of a yeast estrogen screen (YES) assay. The Ishikawa cell line bioassay showed higher sensitivity to 17beta-estradiol (median effective concentration [EC50], 10.7 pM) than did the YES assay (EC50, 480 pM). Fractionation of sediment extracts (all samples collected from 5 sites) showed that the nonpolar fraction was poisonous to yeast cells; the estrogenic activity of this fraction, therefore, could not be measured by YES. However, the nonpolar fraction did not kill the Ishikawa cells. The 17beta-estradiol-equivalent values of 15 extracts (3 fractions from each of 5 sediment samples) ranged from 5.7 to 697 pg/g dry weight according to the Ishikawa cell line bioassay. Chemical analysis using gas chromatography-mass spectrometry revealed that the highest concentrations of endocrine-disrupting chemicals were observed at the sampling station near the sewage treatment plant. The results support that the Ishikawa cell line bioassay is suitable for measuring the estrogenic activity of sediment samples.     

Comparison of the estrogenic activities of seawater extracts from Suruga Bay, Japan, based on chemical analysis or bioassay

The present study compared estrogenicity measured by in vitro bioassay and estrogenicity estimated by the chemical analysis of seawater from Suruga Bay, Japan. Nonylphenol, bisphenol A, estrone, 17beta-estradiol, nonhydroxy polycyclic aromatic hydrocarbons, and hydroxy polycyclic aromatic hydrocarbons, some of which show estrogenic activity, were selected as the target compounds. The yeast two-hybrid system was used to evaluate the estrogenic activities of seawater and chemicals with or without rat liver S9. Concentrations of estrogenic compounds in seawater were measured by chemical analysis using gas chromatography/ mass spectrometry. The main estrogenic compounds in seawater were estrone (< or = 9.2 ng/L), bisphenol A (< or = 1,070 ng/L), and nonylphenol (< or = 276 ng/L). The highest estrogenic activities in seawater were observed near a sewage treatment plant, but the predicted potencies based on the chemistry data were higher than those observed experimentally for the estrogenic activity in seawater. The estrogenicity measured by bioassay was raised considerably after S9 treatment; this observation was limited to the zone of freshwater immediately adjacent to the wastewater outfall.     

Analysis of trace organic contaminants in sediment,pore water,and water samples from Onsan Bay,Korea: instrumental analysis and in vitro gene expression assay

Persistent organic pollutants and alkylphenols (APs) were determined in sediment and water samples from Onsan Bay, Korea, by using instrumental analysis and in vitro gene expression cell bioassay. Polycyclic aromatic hydrocabons (PAHs) were the predominant compounds in sediments with concentrations as great as 573 ng/g dry weight. The PAH concentrations were elevated in sediment from inland rivers that flow through Onsan City (mean: 116 ng/g dry wt) and discharge into Onsan Bay. Concentrations of polychlorinated biphenyls (PCBs) in sediments ranged from <1.00 to 56.2 ng/g dry weight. Among different organochlorine (OC) pesticides analyzed (hexachlorobenzene, hexachlorocyclohexanes, chlordanes, and DDTs), DDT concentrations were the greatest, ranging from <0.01 to 7.58 ng/g dry weight. The spatial gradient of contaminant concentrations suggested that streams and rivers are the major sources of PCBs, PAHs, and APs to the bay. Maximum concentrations of nonylphenol, octylphenol, and bisphenol A in sediments were 860, 11, and 204 ng/g dry weight, respectively. Screening of Onsan Bay sediment samples for dioxinlike activity with the H4IIE-luc in vitro cell bioassay revealed that 17 of 22 samples contained significant dioxinlike activity. Further fractionation of sediment extracts indicated that mid-polar and more polar fractions were responsible for the significant dioxinlike activity. Based on a mass balance analysis, PAHs apparently accounted for only a small portion of dioxinlike responses elicited by sediment extracts. Only one raw extract of sediment elicited a significant estrogenic response by MVLN cells. The combination of instrumental analysis and in vitro bioassay was useful to assess sediment quality and characterize the causative agents or potential toxic compounds present.     

Bioavailability and biodegradation of nonylphenol in sediment determined with chemical and bioanalysis

The surfactant nonylphenol (NP) is an endocrine-disrupting compound that is widely spread throughout the environment. Although environmental risk assessments are based on total NP concentrations, only the bioavailable fraction possess an environmental risk. The present study describes the bioavailability and biodegradability of NP over time in contaminated river sediment of a tributary of the Ebro River in Spain. The bioavailable fraction was collected with Tenax TA(R) beads, and biodegradation was determined in aerobic batch experiments. The presence of NP was analyzed chemically using gas chromatography-mass spectrometry and indirectly as estrogenic potency using an in vitro reporter gene assay (ER(alpha)-luc assay). Of the total extractable NP in the sediment, 95%+/-1.5% (mean +/- standard error) desorbed quickly into the water phase. By aerobic biodegradation, the total extractable NP concentration and the estrogenic activity were reduced by 97%+/-0.5% and 94%+/-2%, respectively. The easily biodegradable fraction equals the potential bioavailable fraction. Only 43 to 86% of the estrogenic activity in the total extractable fraction, as detected in the ER(alpha)-luc assay, could be explained by the present NP concentration. This indicates that other estrogenic compounds were present and that their bioavailability and aerobic degradation were similar to that of NP. Therefore, we propose to use NP as an indicator compound to monitor estrogenicity of this Ebro River sediment. To what extent this conclusion holds for other river sediments depends on the composition of the contaminants and/or the nature of these sediments and requires further testing.     

In Vitro Bioassay Determination of Dioxin-Like and Estrogenic Activity in Sediment and Water from Ulsan Bay and Its Vicinity, Korea

Extracts of sediment and water samples collected from Ulsan Bay, Korea, were screened for their ability to induce dioxin-like and estrogenic gene expression in vitro. Each sample was tested as raw extract (RE) and fractionated extract (FE). Based on the initial screening of RE, 23 of 31 sediment samples showed significant dioxin-like activity in H4IIE-luc bioassay, whereas most sediment samples did not elicit estrogenic response in MVLN bioassay. Most of the activities associated with FE samples revealed that mid-polar (F2) and most polar (F3) fractions were responsible for the significant reporter gene expression in H4IIE-luc bioassay. The results suggest that complex interactions may have depressed the activities of the known arylhydrocarbon receptor (AhR) agonists present in F1 samples. The F2 samples were the most active fraction. All F2 samples except one induced significant dioxin-like activity, and over half of the F2 samples induced significant estrogenic activity. Ten of the F2 samples produced magnitudes of response in H4IIE-luc bioassay similar to those induced by a 2,3,7,8-tetrachlorodibenzo-p-dioxin standard. Sediment associated with F2 samples was estimated to contain 24.9–826 pg TCDD-EQ/g DW. Based on a qualitative mass balance analysis, polycyclic aromatic hydrocarbons (PAHs) appeared to account for both the estrogenic and dioxin-like responses observed. Over half of the F3 samples were either cytotoxic or caused morphological changes in both H4IIE-luc and MVLN cells. Known concentrations of alkylphenols and bisphenol A were not great enough to account for both the estrogenic response and cytotoxicity observed for F3 samples. Despite the apparent toxic or stressful effects, most of F3 samples induced significant dioxin-like activity in vitro, adding to a growing body of evidence that suggests the presence of unidentified, relatively polar, AhR agonists in sediment from some areas. Received: 11 June 2000/Accepted: 28 August 2000     

Horizontal and Vertical Distribution of Estrogenic Activities in Sediments and Waters from Tokyo Bay, Japan

Endocrine-disrupting chemicals with estrogenic activity (e.g., alkylphenols) have been detected in coastal Japan. We aimed to determine estrogenic activity in extracts of river water, seawater, sediments, and sediment cores from Tokyo Bay by in vitro gene expression assay. Fifty-one of 57 extracts had some estrogenic activity. E₂ equivalents (ng E₂ equivalents per gram dry weight or per liter above the limit of detection) in river water samples ranged from 0.70 to 4.01 ng/L; in seawater samples from 0.34 to 2.52 ng/L; and in surface sediments from 2.07 to 12.1 ng/g. The relationship between salinity and estrogenic activity in water samples suggested that fresh water is one source of environmental estrogens in Tokyo Bay. Fractionation of sediment extracts showed that the highest estrogenic activity was observed in the midpolar fraction. The observed activities were compared with activities mediated by known concentrations of nonylphenol, bisphenol-A, estrone, and 17-estradiol. In sediment collected near the sewage treatment plants, the estrogenic activity of the midpolar fraction could be explained about 34% by nonylphenol and estrone contained in this fraction. Core sediment measurements detected estrogenic activity from as far back as the 1960s. The regulations on the industrial wastewater in early 1970s would be one of the main reasons for the lower estrogenic activity in the upper section of the sediment core. The high estrogenic activities as measured in water and sediment samples from Tokyo might be restricted to certain coastal areas.     

In vivo bioassay-guided fractionation of marine sediment extracts from the Southern California Bight, USA, for estrogenic activity

The exposure and uptake of environmental estrogenic compounds have been reported in previous studies of demersal flatfish species in the central Southern California Bight (SCB), USA. The objective of this study was to evaluate the estrogenic or feminizing activity of marine sediments from the SCB by using in vivo vitellogenin (VTG) assays in male or juvenile fish. In 2003, sediments were collected near wastewater outfalls serving the counties of Los Angeles (LACSD) and Orange (OCSD), and the city of San Diego (SD), California, USA. Cultured male California halibut (CH; Paralichthys californicus) were either directly exposed to sediments for 7 d or treated with two intraperitoneal injections of sediment extract over 7 d. The 17beta-estradiol (E2) equivalent values ranged from 1 to 90 microg/kg with LACSD > SD > OCSD. Measurable concentrations of E2 were observed in all sediment extracts and ranged from 0.16 to 0.45 ng/g. Estrone (El) was only observed in sediments near the LACSD outfall (0.6 ng/g). Alkylphenols and alkylphenol ethoxylates were observed in all sediment samples, but were highest near the OCSD outfall, where concentrations of nonylphenol were 3,200 ng/g. Fractionation studies of the LACSD sediment extract collected in 2004 failed to demonstrate relationships between VTG expression and 62 analytes, including E2, which was observed in the whole extract (2.9 ng/g). Oxybenzone (1.6 ng/g) was identified in bioactive fractions as well as unknown compounds of relatively high polarity. These results indicate that estrogen receptor-based assays may underestimate environmental estrogenic activity and estrogenic compounds other than classic natural and xenoestrogens may contribute to estrogenic activity of sediments from the SCB.     

Identification of estrogenic compounds in wastewater effluent

In order to identify the dominant contributors to estrogenic activity in environmental waters, a comprehensive fractionation method using silica gel column chromatography, combined with recombinant yeast assay for detecting estrogenic activity and with gas chromatography-mass spectrometry for quantifying endocrine disruptors and natural estrogens, was developed. The method was applied to the municipal sewage treatment plant (STP) secondary effluent discharged to the Tamagawa River in Tokyo, Japan, where endocrine disruption was observed in wild carp. The instrumental analysis demonstrated that averaged concentrations of nonylphenol, bisphenol A, estrone (E1), and 17beta-estradiol (E2) were 564 +/- 127, 27 +/- 19, 33 +/- 11, and 4.6 +/- 3.0 ng/L, respectively. Based on the concentration and relative potency of these compounds, the natural estrogens E1 and E2 represented more than 98% of the total estrogen equivalent concentration (EEQ) in the STP effluent, while the contribution of phenolic compounds to total EEQ was less than 2%. Estrogenic activities associated with the dissolved phase of the effluent samples were detected by a recombinant yeast assay. By using silica gel column chromatography, the dissolved phase was separated into several fractions that were subjected to the bioassay. The polar fractions exhibited estrogenic activity. The greatest estrogenic activity was found in a polar fraction containing E1 and E2 and represented 66 to 88% of the total estrogenic activities estimated from the bioassay data. These results lead to the conclusion that E1 and E2 were the dominant environmental estrogens in the STP effluent, but a significant contribution to estrogenic activities stems from unidentified components in the effluents.     

Impact of polychlorinated biphenyls contamination on estrogenic activity in human male serum

Polychlorinated biphenyls (PCBs) are thought to cause numerous adverse health effects, but their impact on estrogen signaling is still not fully understood. In the present study, we used the ER-CALUX bioassay to determine estrogenic/antiestrogenic activities of the prevalent PCB congeners and PCB mixtures isolated from human male serum. The samples were collected from residents of an area with an extensive environmental contamination from a former PCB production site as well as from a neighboring background region in eastern Slovakia. We found that the lower-chlorinated PCBs were estrogenic, whereas the prevalent higher-chlorinated PCB congeners 138, 153, 170, 180, 187, 194, 199, and 203, as well as major PCB metabolites, behaved as antiestrogens. Coplanar PCBs had no direct effect on estrogen receptor (ER) activation in this in vitro model. In human male serum samples, high levels of PCBs were associated with a decreased ER-mediated activity and an increased dioxin-like activity, as determined by the DR-CALUX assay. 17beta-Estradiol (E2) was responsible for a major part of estrogenic activity identified in total serum extracts. Significant negative correlations were found between dioxin-like activity, as well as mRNA levels of cytochromes P450 1A1 and 1B1 in lymphocytes, and total estrogenic activity. For sample fractions containing only persistent organic pollutants (POPs), the increased frequency of antiestrogenic samples was associated with a higher sum of PCBs. This suggests that the prevalent non-dioxin-like PCBs were responsible for the weak antiestrogenic activity of some POPs fractions. Our data also suggest that it might be important to pay attention to direct effects of PCBs on steroid hormone levels in heavily exposed subjects.     

Chemical and biological analysis of endocrine-disrupting hormones and estrogenic activity in an advanced sewage treatment plant

The steroid hormones estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)), 17alpha-ethinylestradiol (EE(2)), and their conjugated forms were surveyed throughout an advanced sewage treatment plant (STP). The estrogen concentrations in water and sludge samples, collected in October 2004 and April 2005, were determined by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Simultaneously, the estrogenic activity was quantified using estrogen-responsive reporter cell lines (MELN) to investigate the behavior of overall estrogenic compounds. The estrogen concentrations in the inlet ranged from 200 to 500 ng/L, with the contribution of conjugated forms being higher than 50%. The major estrogens in influent were E(1) and E(3). The estrogenic activity was between 25 and 130 ng/L of E(2) equivalents (EEQs). Estrogen concentrations and estrogenicity measured in the inlet and in primary treated sewage were similar, showing a weak impact of primary treatment on hormone removal. In contrast, both estrogen concentration and estrogenicity decreased during biological treatment, with high removal efficiencies (>90%). Estrone, E(2), and EE(2) persisted in the treated water below 10 ng/L, whereas the estrogenicity was lower than 5 ng/L of EEQs. Estrogen mass flux in the effluent and sludge represented less than 2 and 4%, respectively, of the inlet. Consequently, the fraction of estrogens sorbed into the sludge was very small, and biodegradation was the main vehicle for estrogen elimination. This dual approach, comparing chemical and biological analysis, allowed us to confirm that most of the estrogenic activity occurring in this STP, which receives mainly domestic sewage, resulted from sex hormones.     

Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption

In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.     

The implementation of a battery of in vivo and in vitro bioassays to assess river water for estrogenic endocrine disrupting chemicals

Previous research has shown that accurate evaluation of environmental water samples for estrogenic activity requires a panel of in vitro and in vivo bioassays, which are based on different molecular and cellular action mechanisms. In the current study, a test battery containing four assays was used to analyze water from the Eerste River, South Africa for estrogenicity. Three sites were used for analysis, namely Jonkershoek (control site situated in the mountains at the origin of the Eerste River), sewage effluent from Stellenbosch sewage treatment works and Spier site (sampling site on the Eerste River downstream from Stellenbosch). Estrogenicity was determined using an estrone enzyme linked immuno sorbent assay (ELISA), estrogen induced proliferation of human breast cancer adenocarcinoma cells (MCF-7) also known as the E-SCREEN, estrogen induced suppression of estrogen receptor alpha protein expression (ER-α) in MCF-7 cells (ERα assay) and by monitoring estrogen induced vitellogenin (VTG) synthesis in juvenile Oreochromis mossambicus (VTG assay). Low concentrations of estrone (ranging between 1.4 and 2.2 ng/l) near the detection limit of the assay were detected in samples collected from Jonkershoek. Water from this site shows no estrogenicity in the E-SCREEN, ERα assay or VTG synthesis bioassay. The estrone concentrations in the sewage effluent extracts, as well as Spier site extracts, ranged between 14.7 and 19.4 ng/l. The assays using ERα induction by the MCF-7 cell line, MCF-7 proliferation and in vivo VTG synthesis by juvenile tilapia showed that these samples are estrogenic. The results obtained for the assays in the battery are comparable.     

Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay

The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds.     

A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff

Spreading liquid manure on agricultural fields is a routine way of disposing of animal manure and optimizing the use of nutrients for crops. Limited studies suggest that these wastes may contain a variety of endocrine-disrupting compounds (EDCs) that may be released into aquatic environments through runoff. The purpose of this study was to apply a toxicity identification and evaluation approach to isolate and identify estrogenic compounds in hog manure. A recombinant yeast estrogen screen bioassay was used to detect estrogenicity of high-performance liquid chromatography--separated hog manure fractions. Further analytical analyses of the fractions and comparison to authentic standards resulted in the identification of the endogenous estrogens 17 beta-estradiol (E2) and estrone, and the phytoestrogen metabolite, equol. High levels of equol (6.9-16.6 ppm) were found to be present in manure that was stored for several months. The endocrine-disrupting potential of equol was characterized further by using fish hormone estrogen receptor (ER), sex hormone binding protein (SSBP), and goldfish androgen receptor (AR) radioligand binding assays. Equol was found to be approximately 1,000- and 200-fold less potent that E2 in competing for binding sites of the SSBP and ER, respectively. Equol's potency was 2,200-fold less than testosterone for the AR. Additional studies confirmed the presence of compounds with estrogenic activity in tile drain water after application of hog manure to an agriculture field. In this case, the contribution of equol to the total estrogenicity of the tile drain water was minimal relative to that of natural estrogens. Overall, this study indicates that further work is warranted to assess the impact that EDCs that originate from agricultural runoff may have on the ecology or physiology of exposed biota.     

Influence of 17β-estradiol binding by dissolved organic matter isolated from wastewater effluent on estrogenic activity

The aims of this study were to determine the sorption coefficient (Log K_DOC) of 17β-estradiol (E2), according to the size and composition of dissolved organic matter (DOM) isolated from wastewater effluent using a fluorescence quenching (FQ) method, and to measure the estrogenic changes due to the extent of E2 sorption onto effluent DOM (EfOM) by using an E-screen bioassay. The highest log K_DOC of E2 for a DOM size fraction less than 0.2 μm was observed by 4.87±1.87 L kg⁻¹_DOC, and its estrogenicity was the lowest among all the size fractions at 1.2 ng-EEQ L⁻¹. However, E2 estrogenicity for a DOM size fraction less than 5 kDa was as high as that for the positive control due to the absence of fulvic acid- and protein-like DOM to bind with E2. The estrogenic activity for the hydrophobic fraction bound with E2 was significantly reduced to 1.6 ng-EEQ L⁻¹, while that for the hydrophilic fraction having no binding with E2 was 2.6 ng-EEQ L⁻¹, which was similar to that of the positive control (i.e., 2.8 ng-EEQ L⁻¹). The results support a conclusion that the change in estrogenicity was due to the strong sorption affinity of E2 onto DOM.     

Human colon microbiota transform polycyclic aromatic hydrocarbons to estrogenic metabolites

Ingestion is an important exposure route for polycyclic aromatic hydrocarbons (PAHs) to enter the human body. Although the formation of hazardous PAH metabolites by human biotransformation enzymes is well documented, nothing is known about the PAH transformation potency of human intestinal microbiota. Using a gastrointestinal simulator, we show that human intestinal microbiota can also bioactivate PAHs, more in particular to estrogenic metabolites. PAH compounds are not estrogenic, and indeed, stomach and small intestine digestions of 62.5 nmol naphthalene, phenanthrene, pyrene, and benzo(a)pyrene showed no estrogenic effects in the human estrogen receptor bioassay. In contrast, colon digests of these PAH compounds displayed estrogenicity, equivalent to 0.31, 2.14, 2.70, and 1.48 nmol 17alpha-ethynylestradiol (EE2), respectively. Inactivating the colon microbiota eliminated these estrogenic effects. Liquid chromatography-mass spectrometry analysis confirmed the microbial PAH transformation by the detection of PAH metabolites 1-hydroxypyrene and 7-hydroxybenzo(a)pyrene in colon digests of pyrene and benzo(a)pyrene. Furthermore, we show that colon digests of a PAH-contaminated soil (simulated ingestion dose of 5 g/day) displayed estrogenic activity equivalent to 0.58 nmol EE2, whereas stomach or small intestine digests did not. Although the matrix in which PAHs are ingested may result in lower exposure concentrations in the gut, our results imply that the PAH bioactivation potency of colon microbiota is not eliminated by the presence of soil. Moreover, because PAH toxicity is also linked to estrogenicity of the compounds, the PAH bioactivation potency of colon microbiota suggests that current risk assessment may underestimate the risk from ingested PAHs.     

Assessment of toxicological profiles of the municipal wastewater effluents using chemical analyses and bioassays

The hazardous chemical contamination of untreated wastewater and secondary effluent from the wastewater treatment plant (WWTP) of the city of Zagreb, Croatia was comprehensively characterized using large-volume solid-phase extraction (SPE) and silica gel fractionation, followed by a detailed analysis of the resulting extracts by a combination of chemical and bioassay methods. Over 100 individual contaminants or closely related-contaminant groups were identified by high-resolution gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF). Ecotoxicity profiling of the investigated samples, including cytotoxicity, chronic toxicity and EROD activity; inhibition of the multixenobiotic resistance (MXR), genotoxicicity and estrogenic potential, revealed the most significant contribution of toxic compounds to be present in polar fractions. Wastewater treatment using conventional activated sludge process reduced the initial toxicity of raw wastewater to various extents, ranging from 28% for algal toxicity to 73.2% for an estrogenic activity. The most efficient toxicity removal was observed for the polar compounds.     

某自来水厂水源水有机提取物的雌激素样活性研究

[目的]了解某自来水厂水源水有机提取物的雌激素样活性。[方法]用固相萃取法富集水样,分低、中、高剂量染毒组（体外试验为每毫升染毒量分别相当于10、100、1000mL水源水中有机提取物的剂量;体内试验为每千克体重灌胃量相当于2、5、10、25L水源水中有机提取物的剂量）、阴性对照组和雌二醇阳性对照组,通过体外人乳腺癌（MCF-7）细胞增殖试验、报告基因试验和初断乳大鼠子宫增重试验对水源水有机提取物的雌激素样活性进行研究。[结果]体外细胞增殖试验中,高剂量组较阴性对照组出现细胞增殖效应（P〈0．05）,细胞增殖率为（130．85±13．68）％;而报告基因试验中,中剂量组检测到荧光强度增加（P〈0．05）,相对荧光强度为（207．67±12．40）％。子宫增重试验中,相当于低、中、高剂量染毒组大鼠子宫湿重和黏液重脏器系数与阴性对照组相比差异无统计学意义。[结论]体外试验表明水源水中含有雌激素样活性物质,但体内试验尚不能确定该水源水具有明确的雌激素样活性。     

In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PDBEs, and polybrominated bisphenol A compounds

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as additive flame retardants in plastics and textile materials. PBDEs are persistent compounds and have been detected in wildlife and in human adipose tissue and plasma samples. In this study, we investigated the (anti)estrogenic potencies of several PBDE congeners, three hydroxylated PBDEs (HO-PBDEs), and differently brominated bisphenol A compounds in three different cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression. In human T47D breast cancer cells stably transfected with an estrogen-responsive luciferase reporter gene construct (pEREtata-Luc), 11 PBDEs showed estrogenic potencies, with concentrations leading to 50% induction (EC(50)) varying from 2.5 to 7.3 microM. The luciferase induction of the most potent HO-PBDE [2-bromo-4-(2,4,6-tribromophenoxy)phenol] exceeded that of estradiol (E(2)), though at concentrations 50,000 times higher. As expected, brominated bisphenol A compounds with the lowest degree of bromination showed highest estrogenic potencies (EC(50) values of 0.5 microM for 3-monobromobisphenol A). In an ER alpha-specific, stably transfected human embryonic kidney cell line (293-ER alpha-Luc), the HO-PBDE 4-(2,4,6-tribromophenoxy)phenol was a highly potent estrogen with an EC(50) < 0.1 microM and a maximum 35- to 40-fold induction, which was similar to E(2). In an analogous ER beta-specific 293-ER betas-Luc cell line, the agonistic potency of the 4-(2,4,6-tribromophenoxy)phenol was much lower (maximum 50% induction compared to E(2)), but EC(50) values were comparable. These results indicate that several pure PBDE congeners, but especially HO-PBDEs and brominated bisphenol A-analogs, are agonists of both ER alpha and ER beta receptors, thus stimulating ER-mediated luciferase induction in vitro. These data also suggest that in vivo metabolism of PBDEs may produce more potent pseudoestrogens.     

Specific in vitro toxicity of crude and refined petroleum products: 3. Estrogenic responses in mammalian assays

Current petroleum risk assessment considers only narcosis as the mode of action, but several studies have demonstrated that oils contain compounds with dioxin-like, estrogenic or antiestrogenic, and androgenic or antiandrogenic activities. The present study is the third in a series investigating the specific toxic effects of 11 crude oils and refined products. By employing recombinant mammalian cells stably transfected with the human estrogen receptor alpha (ERα) or beta (ERβ), and expressing the luciferase protein (ERα-U2OS-Luc and ERβ-U2OS-Luc assay), the estrogenicity or antiestrogenicity of oils was studied. All oils, except for two refined oils and one crude oil, induced estrogenic responses. The calculated estrogenic potencies of the oils were six to nine orders of magnitude lower than the potency of 17β-estradiol (E2). Upon coexposure to a fixed concentration of E2 and increasing concentrations of oils, additive, antagonistic, and synergistic effects were revealed. One nautical fuel oil was tested in the human breast carcinoma cell line MCF-7, in which it induced cell proliferation up to 70% relative to the maximal induction by E2. At its minimum effect concentration of 25?mg/L, the oil was also capable of inducing mRNA expression of the estrogen-dependent protein pS2 by a factor of two. The present results indicate that oils naturally contain potentially endocrine-disrupting compounds that are able to influence the estrogenicity of other compounds and may cause biological responses beyond receptor binding.