Effect of diet on fecal and urinary estrogenic activity

The United States Environmental Protection Agency has identified estrogens from animal feeding operations as a major environmental concern, but few data are available to quantify the excretion of estrogenic compounds by dairy cattle. The objectives of this study were to quantify variation in estrogenic activity in feces and urine due to increased dietary inclusion of phytoestrogens. Ten Holstein heifers were assigned to 2 groups balanced for age and days pregnant; groups were randomly assigned to treatment sequence in a 2-period crossover design. Dietary treatments consisted of grass hay or red clover hay, and necessary supplements. Total collection allowed for sampling of feed refusals, feces, and urine during the last 4 d of each period. Feces and urine samples were pooled by heifer and period, and base extracts were analyzed for estrogenic activity (estrogen equivalents) using the yeast estrogen screen bioassay. Feces and urine samples collected from 5 heifers were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify excretion of 7 phytoestrogenic compounds. Excretion of 17-β estradiol equivalents in urine was higher and tended to be higher in feces for heifers fed red clover hay (84.4 and 120.2 mg/d for feces and urine, respectively) compared with those fed grass hay (57.4 and 35.6 mg/d). Analysis by LC-MS/MS indicated greater fecal excretion of equol, genistein, daidzein, coumestrol, and formononetin by heifers fed red clover hay (1634, 29.9, 96.3, 27.8, and 163 mg/d, respectively) than heifers fed grass hay (340, 3.0, 46.2, 8.8, and 18.3 mg/d, respectively). Diet had no effect on fecal biochanin A or 2-carbethoxy-5, 7-dihydroxy-4’-methoxyisoflavone. Four phytoestrogens were detected in urine (2-carbethoxy-5, 7-dihydroxy-4’-methoxyisoflavone, daidzein, equol, and formononetin) and their excretion was not affected by diet. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation.     

Detection of estrogenic activity in herbal teas by in vitro reporter assays

Herbal teas have become popular as alternatives to caffeinated beverages during past two decades. However, toxicological studies of herbal teas have been limited and the safety of herbal teas thus remains unknown. We focused on the estrogenic activities of herbal teas since some of their ingredients are similar to those used in herbal remedies for menopause relief and therefore contain phytoestrogens. To investigate the potential estrogenic activity of extracts prepared from herbal tea mixtures commercially available and to provide useful information for the safety assessment of those products, we initially screened the estrogenic activity in extracts of 15 different herbal teas by an assay using recombinant yeast cells expressing the human estrogen receptor (YES). A distinct estrogenic activity was thus detected in the ethanolic extracts from four herbal tea mixtures. Licorice root was specified as a ingredient responsible for the estrogenic activity in those extracts. In contrast, the aqueous extracts of all herbal tea mixtures we tested exhibited distinct estrogenic activity in YES, thus suggesting the existence of various ingredients that contain estrogenic constituents extractable with water. Among them, the extract of peppermint tea exhibited the highest estrogenic activity. The estrogenic activity in extracts of herbal tea mixtures and specified ingredients were thereafter confirmed by a reporter assay system using transiently transfected HEK293 cells.     

Research Watch: Nonylphenol in trout

Toxicology.     

Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.     

塑料食品接触材料中壬基酚含量的测定

探讨高效液相色谱法测定塑料食品接触材料中壬基酚含量的可行性。样品用正己烷超声波萃取后,氮吹至干,用甲醇定容后离心,上清液采用高效液相色谱分析,用外标法定量。在本试验条件下,壬基酚线性关系良好,相关系数为0.9992,检出限为0.05mg/kg,回收率为90.0%~93.9%,RSD值为2.12%~3.82%。     

Efficient removal of estrogenic activity during oxidative treatment of waters containing steroid estrogens

The discharge of synthetic and natural steroid estrogens from municipal wastewaters to the aquatic environment has received increased attention because of their potential reproductive effects on fish. Using 17alpha-ethinylestradiol (EE2) as a representative steroid estrogen, several oxidants applied in wastewater treatment (chlorine, bromine, ozone, hydroxyl radical, chlorine dioxide, and ferrate) were shown to selectively and rapidly transform EE2. For typically applied oxidant doses, these transformations occur in the time range of seconds to minutes. The resulting initial transformation products of EE2 exhibit a substantially lower in vitro estrogenic activity (< 13% of EE2). For selected structural derivatives of EE2, a quantitative structure-activity relationship was established between substituents changed on the phenolic moiety and the relative in vitro estrogenic activity. In addition, the initial EE2 transformation products that still exhibit residual estrogenic activity are quickly further transformed by most of the tested oxidants. Therefore, oxidative wastewater treatment may serve as a powerful tool to remove estrogenic activity induced by steroid estrogens.     

食品用塑料包装中壬基酚迁移量的检测

建立高效液相色谱-串联质谱法测定食品用塑料包装中壬基酚的迁移量。样品经迁移试验得食品模拟物,水性模拟物直接上机,油基模拟物经甲醇提取、冷冻离心后取上清液分析,外标法定量。在本试验条件下,壬基酚线性关系良好,6种模拟物的相关系数都达到0.998 5以上,两类模拟物的检出限分别为0.01 mg/L和0.01 mg/kg,回收率为89.3%～94.4%,RSD值为2.76%～3.37%。该法简单、灵敏、准确,适用于食品用塑料包装中壬基酚迁移量的检测。     

Removal of estrogenic activity and formation of oxidation products during ozonation of 17alpha-ethinylestradiol

This study investigated the oxidation of the oral contraceptive 17alpha-ethinylestradiol (EE2) during ozonation. First, the effect of ozone (O3) on the estrogenic activity of aqueous solutions of EE2 was studied using a yeast estrogen screen (YES). It could be shown that O3 doses typically applied for the disinfection of drinking waters were sufficient to reduce estrogenicity by a factor of more than 200. However, it proved impossible to completely remove estrogenic activity due to the slow reappearance of 0.1-0.2% of the initial EE2 concentration after ozonation. Second, oxidation products formed during ozonation of EE2 were identified with LC-MS/MS and GC/MS and the help of the model compounds 5,6,7,8-tetrahydro-2-naphthol (THN) and 1-ethinyl-1-cyclohexanol (ECH), which represent the reactive phenolic moiety and the ethinyl group of EE2. Additionally, oxidation products of the natural steroid hormones 17beta-estradiol (E2) and estrone (E1) were identified. The chemical structures of the oxidation products were significantly altered as compared to the parent compounds, explaining the diminished estrogenic activity after ozonation. Overall,the results demonstrate that ozonation is a promising tool for the control of EE2, E2, and E1 in drinking water and wastewater.     

17 beta-estradiol degradation by TiO2 photocatalysis as a means of reducing estrogenic activity

The degradation of 17 beta-estradiol (E2) in water by TiO2 photocatalysis was investigated; concurrently the estrogenic activity of the treated water was evaluated during the photocatalytic reactions by an estrogen screening assay. As a result, 10(-6) M of E2 was totally mineralized to CO2 in 1.0 g L-1 TiO2 suspension under UV irradiation for 3 h. 10 epsilon-17 beta-Dihydroxy-1,4-estradien-3-one and testosterone-like species were elucidated as intermediate products by GC/MS analysis. The mechanisms of E2 degradation by TiO2 photocatalysis were discussed not only experimentally but also theoretically by calculating the frontier electron densities of the E2 molecule. On the basis of the results obtained, it was concluded that the phenol moiety of the E2 molecule, one of the essential functional groups to interact with the estrogen receptor, should be the starting point of the photocatalytic oxidation of E2. This means that the estrogenic activity should be almost lost concurrently with the initiation of the photocatalytic degradation. Actually, the estrogenic activities of the intermediate products were negligible. TiO2 photocatalysis could be applied to water treatment to effectively remove natural and synthetic estrogens without producing biologically active intermediary products.     

Pesticide residues and estrogenic activity in fruit and vegetables sampled from major fresh produce markets in South Africa

Food is likely to be one of the major pathways through which people are exposed to endocrine-disrupting chemicals. With the exception of residual effects, there are concerns that a number of naturally occurring and synthetic chemicals exert adverse effects upon endocrine systems in wildlife and humans. The current study reports selected pesticide concentrations and the total estrogenic activity of fruit and vegetables using the recombinant yeast oestrogen screen (YES) and T47D-KBluc reporter gene assays. A total of 53 food samples (27 fruit and 26 vegetables) from Johannesburg and Tshwane fresh produce markets (in South Africa) were analysed. Of these, 17 contained one to three different pesticide residues with concentrations ranging between 0.01 and 0.68 mg kg^–1, whereas in the rest of the samples no residues were detected. All pesticides detected except in one sample were below the maximum residue level (MRL), but others were unauthorised for use in specified fruit and vegetables. Estrogenic activity was detected in 26.4% (14 samples) of the samples tested, and the estradiol equivalents ranged from 0.007 to 2 pg g^–1. Although the estrogenic activity was low, it may contribute to adverse health effects. Continuous monitoring for pesticides in fruit and vegetables is important in view of the unauthorised pesticides detected in produce from South Africa and the endocrine-disrupting chemical activity found.     

Screening of Korean edible wild plants for estrogenic activity using MCF-7 cell proliferation assay

This present study was performed to investigate estrogenic activity of Korean edible wild plants for alternative of estrogen replacement therapy in postmenopausal women. When the estrogenic activity was measured by estrogen induced proliferation of MCF-7 cells, of investigated 29 extracts, 3 samples of kudzu vine (Pueraria thunbergiana), Cardamine leucantha, and David Vetchling (Lathyrus davidii) exhibited a significant proliferation of above 20% at concentration of 100 μg/mL. This stimulation of MCF-7 cell proliferation could be completely blocked by addition of estrogen antagonist ICI 182,780 (100 nM), indicating estrogen receptor-dependent mechanism of these estrogenic effects on MCF-7 cell proliferation.     

Nonylphenol in anaerobically digested sewage sludge from New York State

Nonylphenols (NPs) have been identified as xenoestrogens and have been found at high concentrations in Canadian and European biosolids. While nonylphenol polyethoxylates (NPEOs) are being phased out and regulated in several European countries, there is currently no regulation of these compounds in the United States, and little information is available concerning the presence of NPs in U.S. biosolids. Anaerobically digested sewage sludge from five wastewater treatment plants in central New York State was analyzed for the presence of NPs. Samples were taken from treatment plants in both small municipalities and larger metropolitan areas with a range of industrial inputs. Samples were extracted via Soxhlet apparatus and analyzed by GC/MS. The various isomers of NP were summed yielding total NP concentrations as high as 1840 mg/kg with a mean of 1500 mg/kg on a dry weight basis. These values are two to five times as high as previously reported concentrations for U.S. and Canadian biosolids from plants using similar treatment schemes.     

建议皮革工业禁用烷基酚聚氧乙烯醚

烷基酚聚氧乙烯醚（APE）是城市污水的主要污染物之一，在APE的自然降解过程中会产生壬基酚（NP）、短链乙氧基化物和相应的氧化物，如壬基酚乙二醇单醚（NPIE）、壬基酚二乙二醇单醚（NP2E）、壬基酚氧基乙酸（NP2EC）和壬基酚氧基乙氧基乙酸（NP2EC）等。这些产物，特别是NP、NPIE和N2PE的毒性和雌激素活性明显、生物降解性差、亲脂性强、易在污泥和生物体内积累，并可能通过食物链以慢性作用危及所有物种的生存，因此，禁用烷基酚聚氧乙烯醚是减轻环境恶化、保护生态系统的必要措施之一。     

Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed.

Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor alpha (hERalpha) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens. In the present study this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in animal feed. This validation was performed according to EC Decision 2002/657. Twenty blank animal feed samples, including milk replacers and wet and dry feed samples, were spiked with 17beta-estradiol (E2beta) at 5 ng g(-1), 17alpha-ethynylestradiol (EE2) at 5 ng g(-1), diethylstilbestrol (DES) at 10 ng g(-1), zearalenone at 1.25 microg g(-1) or equal at 200 microg g(-1). All of these blank and low estrogen spiked feed samples fulfilled the CCalpha and CCbeta criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CCalpha and were thus classified as compliant, and at least 19 out of the 20 spiked samples gave a signal above this CCalpha (beta = 5%) and were thus classified as suspect. The method was specific and estrogens in feed were stable for up to 98 days. In this study we also present long-term performance data and several examples of estrogens found in the routine screening of animal feed. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed.     

Enantioselectivity in estrogenic potential and uptake of bifenthrin

Despite the fact that the biological processes of chiral compounds are enantioselective, the endocrine disruption activity and uptake of chiral contaminants with respect to enantioselectivity has so far received limited research. In this study, the estrogenic potential and uptake of the enantiomers of a newer pyrethroid insecticide, bifenthrin (BF), were investigated. Significant differences in estrogenic potential were observed between the two enantiomers in the in vitro human breast carcinoma MCF-7 cell proliferation assay (i.e., the E-SCREEN assay) and the in vivo aquatic vertebrate vitellogenin enzyme-linked immunosorbent assay (ELISA). In the E-SCREEN assay, the relative proliferative effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2% and 20.9%, respectively, and the relative proliferative potency ratios were 10% and 1%, respectively. The cell proliferation induced by the two BF enantiomers may be through the classical estrogen response pathway via the estrogen receptor (ER), as the proliferation induced by the enantiomers could be completely blocked when combined with 10-6 mol/L of the ER antagonist ICI 182,780. Measurement of vitellogenin induction in Japanese medaka (Oryzias latipes) showed that, at an exposure level of 10 ng/mL, the response to 1S-cis-BF was about 123 times greater than thattothe Renantiomer. Significant selectivity also occurred in the uptake of BF enantiomers in the liver and other tissues of J. medaka. These results together suggest that assessment of the environmental safety of chiral insecticides should consider enantioselectivity in acute and chronic ecotoxicities such as endocrine disruption.     

Biological validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens

The combined estrogenic effects of mixtures of environmental pollutants in the in vitro ER-CALUX (chemical activated luciferase gene expression) bioassaywere examined to biologically validate a sample preparation method for the analysis of estrogenic compounds in sediment. The method used accelerated solvent extraction (ASE) and gel permeation chromatography (GPC) and was validated with respect to recovery of biological response taking mixture effects into account. Four mixtures of three to six xenoestrogenic compounds (bisphenol A, 4-nonylphenol, (4,4'-dichlorodiphenyl)trichloroethane, (2,4'-dichlorodiphenyl)trichloroethane, dieldrin, 4-n-octylphenol, alpha-chlordane, dibutylphthalate, (4,4'-dichlorodiphenyl)dichloroethylene, and 2,4,5-trichlorobiphenyl) were prepared. Experimentally determined mixture effects were well described by the concept of concentration addition (CA), as expected for similarly acting compounds. Observed estradiol equivalence factors of the mixtures (on average 1.2 +/- 0.3) agreed very well with the value predicted according to CA. The sample preparation method was then applied to pure mixtures of standards and to sediment spiked with one of the mixtures. Recoveries of estrogenic compounds were estimated by determination of their mixture potencies in ER-CALUX and compared to the mixture effects predicted by CA. Recoveries of estrogenic activity were between 80 and 129%, indicating that the additive behavior of mixtures of xeno-estrogens is well conserved during sample preparation. Together with an average repeatability of 18.3%, low average limit of detection (2.6 +/- 1.8 pg of EEQ/ g), and coefficient of variance (3.5 +/- 3.3%),this demonstrated the suitability of the sample preparation method for the analysis of mixtures of (xeno-)estrogenic compounds in sediment with the ER-CALUX assay.     

Transformation of oxidation products and reduction of estrogenic activity of 17beta-estradiol by a heterogeneous photo-Fenton reaction

A novel photo-Fenton catalyst, alpha-FeOOH loaded resin (alpha-FeOO HR), was synthesized and evaluated through transformation of a steroidal endocrine disrupting compounds (EDC), 17beta-estradiol (E2), under weak UV irradiation in the presence of H2O2. E2 photodegradation intermediates elucidated in detail by GC/MS and LC/MS/MS analyses and detailed reaction pathways are proposed. A yeast-based estrogen screen for E2 and its photodegradation intermediates was performed to measure the reduction of estrogenic activity in different water matrices during the heterogeneous photo-Fenton process. The results showed that alpha-FeOOHR not only degraded E2 but also removed the estrogenic activity originating from E2, its degradation intermediates, and its products. However, the water matrix present in drinking water may impactestrogenic activity reduction. The results are important to evaluate the ability of photo-Fenton advanced oxidation processes in reducing EDCs and their associated estrogenicity from drinking water.