MECHANISMS IN ENDOCRINOLOGY: Estrogens in consumer milk: is there a risk to human reproductive health?

Possible effects of xenoestrogens on human health, in particular on male reproductive health, have attracted considerable attention in recent years. Cow's milk was suggested in numerous publications as one of possible sources of xenoestrogens that could affect human health. Although milk has undoubtedly many beneficial health effects and could even have a role in reducing incidence of some cancers, concerns were raised about presumably high levels of estrogens in cow's milk. In intensive farming, concentrations of estrogens in milk are higher due to long milking periods that today extend long into the pregnancy, when concentrations of estrogens in the cow's body rise. Numerous studies examined potential effects of milk on reproductive health and endocrine-related cancers in both experimental studies with laboratory animals, and in human epidemiological studies. In the present review article, we compiled a review of recently published literature about the content of estrogens in cow's milk and potential health effects, in particular on reproductive system, in humans. Although results of published studies are not unequivocal, it seems that there is stronger evidence suggesting that amounts of estrogens in cow's milk are too low to cause health effects in humans.

Determination of bisphenols with estrogenic activity in plastic packaged baby food samples using solid-liquid extraction and clean-up with dispersive sorbents followed by gas chromatography tandem mass spectrometry analysis.

Bisphenols (BPs) are a family of chemicals with known endocrine disrupting activity. Bisphenol A (BPA) is the most representative prototype of this group of chemicals. Recently, the use of BPA, a prototype of endocrine disruptors, has been reduced and replaced with structural analogs due to its negative effects on both the environment and consumers. In this work, a new method is presented for the determination of seven BPs, with estrogenic activity in ready-to-eat plastic packaged baby foods. The procedure involves the isolation of the analytes using solid-liquid phase extraction with acetonitrile followed by a clean-up step with a mixture of dispersive-SPE sorbents (C18 and PSA) and magnesium sulphate, to reduce matrix effect from proteins, sugars and lipids. Extraction parameters were optimized using multivariate optimization methods. The compounds were detected and quantified by gas chromatography tandem mass spectrometry (GC-MS/MS). The limits of quantification were between 0.1 and 1.2ngg-1 for the studied analytes. The method was validated using matrix-matched calibration and recovery assays with spiked samples. Recovery rates were between 91% and 110% and % RSD was lower than 13% in all cases. The method has been successfully applied for the determination of these endocrine disrupting chemicals (EDCs) in samples of a novel type of food consumed by pre-schoolers. This is the first study to analyze EDCs in plastic packaged foods consumed by this target group.

Rapid and ultrasensitive detection of endocrine disrupting chemicals using a nanosensor-enabled cell-based platform.

Endocrine disrupting chemicals (EDCs) interact with estrogen receptors (ERs), causing a broad range of adverse health effects. Current assays for EDC activity are slow and often lack sensitivity. We report here an ultra-sensitive nanosensor that can detect estrogenic cellular changes in ER(+) MCF-7 cells rapidly (minutes) at several orders of magnitude lower than the generally used assays. Notably, the sensor responses at these ultra-low EDC levels correlated with an increased synthesis phase (S-phase) cell population of EDC-treated cells. The nanosensor was also able to detect binary EDC mixture effects, with synergism observed for bisphenol A (BPA) - 17ß-estradiol (E2), and antagonism for dicyclohexylphthalate (DCHP) - E2 and benzo(a)pyrene (BaP) - E2.

Local effect of bisphenol A on the estradiol synthesis of ovarian granulosa cells from PCOS.

Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50 ± 63.70 pg/ml) was significantly higher than that from non-PCOS patients (338.00 ± 57.88 pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1 µM for 24 h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.

Estrogenic compound profiles in an urbanized industry-impacted coastal bay and potential risk assessment by pollution indices and multivariative statistical methods.

The occurrence and distribution of target estrogenic compounds in a highly urbanized industry-impacted coastal bay were investigated, and contamination profiles were evaluated by estimating total estradiol equivalents (∑EEQs) and risk quotients (RQs). Phenolic compounds were the most abundant xenoestrogens, but seldom showed contribution to the ∑EEQs. The diethylstilbestrol (DES) and 17α-ethinylestradiol (EE2) were the major contributors followed by 17ß-estradiol (E2) in comparison with a slight contribution from estrone (E1) and estriol (E3). Both ∑EEQs and RQs indicated likely adverse effects posed on resident organisms. Further, multivariate statistical method comprehensively revealed pollution status by visualized factor scores and identified multiple "hotspots" of estrogenic sources, demonstrating the presence of complex pollution risk gradients inside and particularly outside of bay area. Overall, this study favors the integrative utilization of pollution indices and factor analysis as powerful tool to scientifically diagnose the pollution characterization of human-derived chemicals for better management decisions in aquatic environments.

Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen.

The estrogenicity of waters collected from an important hydrological system in Brazil (Paraiba do Sul and Guandu Rivers) was assessed using the yeast estrogen screen (YES) assay. Sampling was performed in rivers and at the outlets of conventional water treatment plants (WTP). The removal of estrogenic activity by ozonation and chlorination after conventional water treatment (clarification and sand filtration) was investigated employing samples of the Guandu River spiked with estrogens and bisphenol A (BPA). The results revealed a preoccupying incidence of estrogenic activity at levels higher than 1ngL(-1) along some points of the rivers. Another matter of concern was the number of samples from WTPs presenting estrogenicity surpassing 1ngL(-1). The oxidation techniques (ozonation and chlorination) were effective for the removal of estrogenic activity and the combination of both techniques led to good results using less amounts of oxidants.

Simultaneous determination of 30 hormones illegally added to anti-ageing functional foods using UPLC-MS/MS coupled with SPE clean-up.

A novel analytical method employing solid-phase extraction (SPE) coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 30 hormones in anti-ageing functional foods (capsules, powders and tablets). The analytes were extracted with acetic acid-acetonitrile (1-99 v/v), methanol and acetone, respectively. The extract was purified using a combined column, followed by analyte detection with electrospray ionisation in positive- or negative-ion modes. The results indicated that the 30 compounds had good linear correlations in the range of 1-1000 µg kg⁻¹, and the correlation coefficients were above 0.99. The limits of detection (LOD) and limits of quantification (LOQ) were 0.03-2 and 0.1-5 µg kg⁻¹, respectively. The average recovery of 30 compounds at the three spiked levels varied from 74.7% to 124.1%, and the relative standard deviation (RSD) was 2.4-15.0%. This method was applied to the analysis of hormones in 14 real samples of which seven hormones (such as estrone, dienestrol) were detected in four samples, but the remainder of the hormones were not detected. The developed method is sensitive, efficient, reliable and applicable to real samples.

An imprinted crystalline colloidal array chemical-sensing material for detection of trace diethylstilbestrol.

Based on a combination of the molecular imprinting technique and polymerized crystalline colloidal array, we have developed an imprinted crystalline colloidal array (ICCA) chemical-sensing material for the real-time and label-free detection of diethylstilbestrol (DES) in aqueous solution. This novel sensing material was prepared by a noncovalent and self-assembly approach using liquid monodispersed DES-imprinted colloidal spheres and was characterized by a three-dimensional (3D) ordered opal structure in which numerous nanocavities were derived from DES imprinting. Thus, the inherent high affinity of the nanocavities allowed ICCA to recognize DES with high specificity, and changes of the ordered periodic structure enabled ICCA to transfer the recognition events into readable optical signals (label-free). Owing to the special opal structure and without interference from the bulk hydrogel film, the ICCA enabled the rapid and sensitive detection of the target analyte. The understanding of the recognizing response has also been advanced by using molecular modeling software to compute rational interaction between the template molecules and the function monomers. After careful optimization of the assay conditions, the ICCA could decrease its diffraction intensity within just 7 min according to the DES concentration from 2 ng mL(-1) to 8.192 µg mL(-1), whereas there were no obvious diffraction intensity changes for the DES analogues. The adsorption results showed that the homogenous structure and large surface area of ICCA could improve its adsorption capacity. Therefore, such a sensing material with high selectivity, high sensitivity, high stability, and easy operation might offer an attractive alternative for establishing optical sensors for the rapid real-time monitoring of different residues in food and the environment.

T-2 toxin, zearalenone and fumonisin B1 in feedstuffs from China.

A total of 420 feedstuff samples were collected from China to determine and quantify T-2 toxin (T-2), zearalenone (ZEN) and fumonisin B1 (FB1). The contents of mycotoxins were determined by reverse-phase high-performance liquid chromatography with three-step cleanup and fluorescence detection. The mean recoveries of mycotoxins in spiked feedstuffs ranged from 72.2% to 95%. The limits of detection and quantification ranged from 1.8 to 5.7 and 6 to 19 µg/kg, respectively. Of the 420 analysed feedstuff samples, the incidence of T-2, ZEN and FB1 was 79.5%, 85.2% and 96.1%, respectively; levels detected ranged from 10-735, 35-1478 and 20-6568 µg/kg, respectively. The results suggest a risk for domestic animals and humans. Further, the procedure was found to be suitable for determination of mycotoxins in feedstuffs and can be used for routine analysis. This is the first report in China on natural contamination of feedstuffs with these mycotoxins.

Application of ethyl chloroformate derivatization for solid-phase microextraction-gas chromatography-mass spectrometric determination of bisphenol-A in water and milk samples.

A simple and rapid analytical method based on in-matrix ethyl chloroformate (ECF) derivatization has been developed for the quantitative determination of bisphenol-A (BPA) in milk and water samples. The samples containing BPA were derivatised with ECF in the presence of pyridine for 20 s at room temperature, and the non-polar derivative thus formed was extracted using polydimethylsiloxane solid-phase microextraction (SPME) fibres with thicknesses of 100 µm followed by analysis using gas chromatography-mass spectrometry. Three alkyl chloroformates (methyl, ethyl and isobutyl chloroformate) were tested for optimum derivatisation yields, and ECF has been found to be optimum for the derivatisation of BPA. Several parameters such as amount of ECF, pyridine and reaction time as well as SPME parameters were studied and optimised in the present work. The limit of detection for BPA in milk and water samples was found to be 0.1 and 0.01 µg L(-1), respectively, with a signal-to-noise ratio of 3:1. The limit of quantitation for BPA in milk and water was found to be 0.38 and 0.052 µg L(-1), respectively, with a signal-to-noise ratio of 10:1. In conclusion, the method developed was found to be rapid, reliable and cost-effective in comparison to silylation and highly suitable for the routine analysis of BPA by various food and environmental laboratories.

Occurrence of estrogenic chemicals in South Korean surface waters and municipal wastewaters.

Broad scale monitoring of estrogenic compounds was performed at 19 sampling points throughout the Yeongsan and Seomjin river basins and 5 wastewater treatment plants (WWTPs) adjacent to the Gwangju area, Korea, from December 2005 to August 2007. The concentrations of estrogenic compounds, including estrone (E1), 17ß-estradiol (E2), 17α-ethynylestradiol (EE2), bisphenol-A, nonylphenol (NP) and 4-octylphenol (OP), in the samples was measured with gas chromatography/mass spectrometry (GC-MS). In addition, the estrogenic activities throughout the river were investigated using the E-screen assay. Of the six estrogenic chemicals, NP (114.6-336.1 ng L(-1)) and EE2 (0.23-1.90 ng L(-1)) were detected at the highest and lowest levels, respectively in both the river waters and the WWTP effluents. Bisphenol-A showed the largest concentration range, from 7.5 to 335 ng L(-1). The concentrations of E1, E2 and octylphenol ranges were 3.6-69.1, 1.2-10.7, and 2.2-16.9 ng L(-1), respectively. According to the calculated estradiol equivalent concentration (EEQ); however, no estrogenic contribution was observed due to the phenolic compounds in the river waters and effluents. E1 and E2 dominated in both the river water and effluent samples, with contributions to the calculated EEQ of over 79 and 77%, respectively. Conversely, EE2 was rarely detected in the river waters (21%) and effluents (0%). The largest contribution of EE2 to the calculated EEQ was 21% in the river water at S-7. The levels of E1, E2, and EE2 were remarkably decreased in the effluents, indicating that the 5 WWTPs did not contribute to the estrogenic effect of the receiving streams. Overall, the WWTPs did not contributed to the estrogenic activity of the receiving waters, but the livestock industry or wildlife may play an important role in the estrogenic contribution to river water.

Gynaecomastia linked to the intake of a herbal supplement fortified with diethylstilbestrol.

This study reports the findings of a supplement marketed on the Internet for prostate problems. The supplement was orally taken by a 60-year-old man with divergent hormonal levels and who was surgically treated for gynaecomastia: development of abnormally large mammary glands in males. The supplement showed a strong effect in a yeast oestrogen bioassay, expressing a yeast-enhanced green fluorescent protein (yEGFP) upon exposure to oestrogens. Using both nuclear magnetic resonance (NMR) and a gradient liquid chromatographic time-of-flight mass spectrometric (LC/TOF-MS) method, the response was shown to be caused by very high levels of diethylstilbestrol, known for causing gynaecomastia. The gynaecomastia was most probably caused by this orally taken 'natural' herbal supplement, as the patient's hormonal levels also returned to normal again when stopping the use of it. This case demonstrates that physicians need to be aware of the use of supplements with illegal components that may be responsible for unwanted side-effects.

The detection of dioxin- and estrogen-like pollutants in marine and freshwater fishes cultivated in Pearl River Delta, China.

In this study we aimed to assess the dioxin- and estrogen-like activities of contaminants extracted from twenty species of freshwater and seawater fishes, using luciferase reporter assays. Transfected MCF7 cells were treated with sample extracts and luciferase activities were then measured at 24-h of post-treatment. The mean values of the detected dioxin- and estrogen-like activities in the freshwater fishes were 25.3 pg TEQ/g ww and 102.3 pM EEQ/g ww whereas in the seawater fishes, the values were 46.2 pg TEQ/g ww and 118.8 pM EEQ/g ww. Using sample-relevant dosage of estrogen, inductions of cell proliferation markers (i.e. retinoblastoma, cyclin D) and stimulations of cell growth were revealed by Western blotting, colony formation and BrdU uptake assays. A cotreatment with TCDD significantly reduced these effects. Using the sample extracts with different dioxin- and estrogen-like activities, similar observation was revealed. The data highlighted the mixture effect of food contaminants on human health.

Understanding the gap between the estrogenicity of an effluent and its real impact into the wild.

To study the reliability between in vitro and in vivo data collected downstream 2 sewage treatment plants (STP) as well as from bleached kraft mill industry (BKME), 5 rivers (3 impacted and 2 references) were investigated in the Walloon region (southern of Belgium). For the in vitro part of the work, water samples were collected to measure the estrogenicity of the 'out' effluent compared to reference sample point by MCF-7 assay. Results indicated significant estrogenicity of effluents from STP and BKME and a weak estrogenicity in reference sites. However, estradiol equivalents (EEQ) estimated into rivers were probably too low to impact wild population. Chemical analysis of 13 compounds of interest indicated that extraction procedure used in this study gave low recoveries of estrogen-like xenobiotics, leading to probably under-estimated MCF-7 responses. Surprisingly, a full scan mode has revealed an unexpected compound in the sample of BKME which was: 7-isopropyl-1,1,4a-trimethyl-1,2,3,4a,9,10,10a-octahydrophenanthrene, a product of pulp mill manufacture. In parallel to in vitro, in vivo assessment of estrogenic impact of effluent was followed on the gudgeon (Gobio gobio). Samples were achieved during 2 different periods of the reproductive cycle, resting period (RP) and pre-spawning period (pSP). Unspecific physiological parameters to estrogenic exposure (gonadosomatic index and systematic testis cell counting) displayed no significant differences related to endocrine disruption of the reproductive tract, only differences were correlated with the reproductive state of fish (RP versus pSP). Concerning the potent biomarker of estrogen exposure, vitellogenin (vtg), only basal induction was revealed but not related to estrogenic exposure. Nevertheless, vtg over-expression was found for male fish presenting a feminization of the reproductive tract captured downstream the STP station of Wégnez in the Vesdre River. Intersexuality, another indicator of the estrogenicity impact in fish, was observed in every site. Actually, ovotestis was systematically formed by protoplasmic oocyte observed in low percentage in every group analysed (impacted and references). Moreover, in fish captured in Wégnez, oocyte diameter was significantly higher compared to the other groups. In this study, only moderate to none impact in population of gudgeon was noticed. Moreover, in this case no discrepancy between in vitro and vivo was viewed although both approaches revealed gaps in monitoring effluent incidence into the environment. We should remain careful in the interpretation when only partial approaches are used in order to characterize impact in the aquatic milieu.

Quantitative screening of stilbenes and zeranol and its related residues and natural precursors in veal liver by gas chromatography-mass spectrometry.

An existing gas chromatography-mass spectrometry-based quantitative screening method for the regulatory analysis of the resorcylic acid lactones zeranol, taleranol, and zearalanone and the stilbene anabolic steroids diethylstilbestrol and dienestrol was extended to include natural precursors of zeranol (zearalenone, alpha-zearalenol, and beta-zearalenol) in veal liver. No changes in sample preparation were required; the instrumental conditions were selected to effect a suitable chromatographic separation and detection of the analytes. Validation experiments were performed to verify the performance and applicability of the extended method for the quantitative screening of the original and additional analytes in veal liver in the concentration range from 0.5 to 2.0 microg/kg. The limits of detection were 0.08-0.19 microg/kg. The limits of quantitation were 0.27-0.64 microg/kg. Recoveries were 29-67%. Combined relative measurement uncertainty estimates were 6-21%.

Zearalenone contamination and the causative fungi in sorghum.

Natural contamination by zearalenone, a toxic metabolite of Fusarium fungi, was surveyed in 160 samples of sorghum imported from 2001 to 2006 into Japan for feed. Of these 160 samples, 84 (52.5%) were contaminated with zearalenone, ranging in concentration from 60 to 7.260 microg/kg. In the contaminated sorghum samples, F. semitectum, F. verticillioides, F. oxysporum, and other Fusarium spp. were detected. The concentration of zearalenone was well correlated with the development of colonies of F. semitectum and other Fusarium spp. When the isolates of F. semitectum and F. verticillioides were cultivated on sorghum, zearalenone was found only in F. semitectum culture. These results indicate that F. semitectum is a causal fungus of zearalenone contamination in sorghum.

Severe abnormalities in the reproductive organs of mice caused by chemical substances contained in heavy oil.

It is well known that heavy oil pollution results in various negative impacts on the marine environment. Although there is a low possibility of direct exposure to heavy oil, the chemical substances contained in heavy oil may be released into the environment and accumulated by marine organisms which in turn can be taken by humans via the food chain. In this study, we examined the biological risk of heavy oil extract using the common mouse, whose genetic backgrounds and immune system are well known and relatively homologous to humans. Water-soluble fraction (WSF) was extracted from heavy oil with water and the extract orally administrated to female or male mice for 7 days. In the WSF administrated group, cystoma-like formation was observed in the ovary in approximately 80% of female mice. On the other hand, we found that the prostate gland size in male mice was markedly reduced in comparison with male control mice. Continuous administration of WSF for 28 days resulted in continued hypertrophy of the cystoma around the ovary and atrophy in the prostate gland. In addition, it was revealed that chemical substances within WSF have estrogenic activity. A major component of heavy oil, polycyclic aromatic hydrocarbons (PAHs), is known to present estrogenic activity. It is likely that the cystoma-like formation in female mice and atrophy of prostate gland in male resulted of estrogenic substances present in the WSF which might be the PAHs.

One-step simultaneous immunochromatographic strip test for multianalysis of ochratoxin a and zearalenone.

Individual immunochromatographic assays (ICG) for ochratoxin A (OTA) and zearalenone (ZEA) were optimized and used in the development of a one-step simultaneous immunochromatographic assay (OS-ICG) for the rapid multianalysis of two mycotoxins in corn samples. The nitrocellulose membrane of the OS-ICG was treated with OTA-bovine serum albumin (BSA), ZEA-ovalbumin (OVA), and anti-mouse IgG in the OTA test, ZEA test, and control zones, respectively. Monoclonal antibody-gold conjugates (OTA3 MAb-gold and ZEA2C5 MAb-gold) were sprayed onto the conjugate pad. The visual detection limits were 2.5 and 5 ng/ml for OTA and ZEA, resepectively, and the results were obtained within 15 min after starting the analysis. An efficient, simple, and rapid extraction method using 30% MeOH/PBS was established and validated by analyzing the corn samples spiked with OTA/ZEA mixtures (0/0, 5/10, 10/20, and 20/30 microg/kg). The cut-off values of the OS-ICG for the spiked corn were 5 and 10 microg/kg for OTA and ZEA, respectively. Natural corn samples were analyzed by OS-ICG, direct competitive enzyme-linked immunosorbent assay (DC-ELISA), and HPLC. Results of the OS-ICG were in good agreement with those obtained by DC-ELISA and HPLC. The developed OS-ICG offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site simultaneous determination of OTA and ZEA in cereals, food, and agricultural products in one analytical cycle.

Detection of mycoestrogen zearalenone by a molecularly imprinted polypyrrole-based surface plasmon resonance (SPR) sensor.

The aim of the present work was to investigate the feasibility of employing the molecular imprinting polymer technique for detecting the mycotoxin zearalenone using a surface plasmon resonance (SPR) transducer. The molecularly imprinted polypyrrole (MIPPy) film was prepared by electropolymerization of pyrrole onto the bare Au chip in the presence of a template zearalenone molecule. The MIPPy-SPR sensor exhibited a linear response in the range of 0.3-3000 ng/mL (R (2) = 0.993) for detection of zearalenone. The selectivity efficiencies of zearalenone and other structurally related analogues were 1.0 and 0.15-0.27, respectively. The limit of detection and average recovery of blank corn matrix spiked with 30 ng/g zearalenone were 0.3 ng/g and 89%, respectively, and these were found to be comparable to those obtained by enzyme-linked immunosorbent assay. These results suggest that a combination of SPR sensing with MIPPy film is a promising alternative method for the detection of zearalenone.

Xenoestrogens: mechanisms of action and some detection studies.

Xenoestrogens are defined as chemicals that mimic some structural parts of the physiological estrogen compounds, therefore may act as estrogens or could interfere with the actions of endogenous estrogens. Two subtypes of the ER are known, the ER alpha and ER beta, and both have a distinct tissue distribution and play a distinct role in physiology. Receptor dimmer assumes a distinctive conformation, binds to its estrogen response element (ERE), interacts with the general transcription complex bound to the TATA box within the respective gene promoter, and regulates gene transcription. The discovery and identification of co-activators and co-repressors provided crucial insights into the ER action. New evidence indicates that the activation of additional transcription factors as well as the action of xenoestrogens through estrogen receptors located outside the cell nucleus (in the plasma membrane, mitochondria and probably the cytosol) should be considered. The levels of exposition to xenoestrogens and the age of the investigated animal can have a significant effect on its development and reproduction. Therefore, several in vivo and in vitro assays have been developed to assess the estrogenic-like activity of individual compounds or natural mixtures. In this review, selected methods applied in physiological studies have been described. One of the most extensively used in vivo assays for estrogenicity is the rodent uterotrophic assay. In order to analyze the estrogenic properties of xenoestrogens, morphological, histological, biochemical and molecular studies should be introduced. A variety of in vitro tests have been established to determine estrogenic potency of xenoestrogens but even a combination of them is not able to predict their actual action in the organism. There is a need for the studies on all potential xenoestrogens to describe tissue-specific activities, and via which pathways in those tissues these compounds either disrupt or mimic hormone action.