Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated with > or =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.     

Magnetic acoustic resonance immunoassay (MARIA): a multifrequency acoustic approach for the non-labelled detection of biomolecular interactions

A unique sensing platform, comprising an electromagnetic field detector and an acoustic resonator, has been used as a wireless system for remote sensing of biorecognition events. The MARS (Magnetic Acoustic Resonator Sensor) technique has proven useful for detecting the formation of protein multilayers derived from specific binding phenomena. The technique enables multifrequency analysis, without the need of electrodes attached to the sensing element, and also facilitates the in situ surface modification of the substrate for antibody attachment. The MARS sensor was utilized as the platform on which a standard immunoassay was carried out. Two different conditions for the attachment of the first antibody to the quartz surface were tested: (i) Adsorption of the antibody onto the surface of a bare quartz disc; (ii) covalent immobilization of the antibody to a chemically modified quartz surface. Both methods can be successfully utilized for the 'label-less' detection of the biorecognition event between goat IgG and anti-goat IgG by analysis of the multifrequency spectrum. Covalent attachment of the primary antibody results in a more efficient immobilization, with higher surface density, and a consistently enhanced response for the binding of the secondary antibody. This approach will be of interest to life scientists and biochemists that require high performance assay methodologies that do not use chemical labels.     

A nonisotopic estrogen receptor-based assay to detect estrogenic compounds

We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17beta-estradiol for binding to the receptor, which leaves 17beta-estradiol free to bind to an anti-17beta-estradiol antibody. Unbound anti-17beta-estradiol antibody then binds to immobilized 17beta-estradiol-protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods.     

A SPR-based immunosensor for the detection of isoproturon

The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.     

GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.     

Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG).

This study reports on some environmental chemicals with estrogenic activity (xenoestrogens) and their binding interaction for human plasma sex-hormone binding globulin (hSHBG). The binding affinity constant of these xenoestrogens was measured in equilibrium conditions by solid phase binding assay, and their ability to displace endogenous testosterone and estradiol from hSHBG binding sites was determined with an ammonium sulfate precipitation assay in native plasma from normal men and women. The data showed that some of these xenoestrogens bind hSHBG, with a reversible and competitive binding activity for both [3H]testosterone and [3H]17beta-estradiol and with no apparent decrease in the number of hSHBG binding sites. Their respective binding affinity constants were low, ranging from 0.02 to 7.8 10(5) 1 x mol(-1). However, in native plasma from normal men and women, they were able to dose-dependently increase concentrations of hSHBG-unbound testosterone and/or estradiol. In this study, 4-nonylphenol and 4-tertoctylphenol, two alkylphenols used as surfactants in many commercial products, and bisphenol A and O-hydroxybiphenyl, widely used in the plastics industry, were identified as potent hSHBG-ligands. Additionally, the flavonoid phytoestrogens genistein and naringenin were also identified as hSHBG ligands, whereas their glucoside derivatives, genistin and naringin, had no binding activity for hSHBG. From these data, it is suggested that hSHBG binding may transport some contaminant xenoestrogens into the plasma and modulate their bioavailability to cell tissues. On the other hand, xenoestrogens may also displace endogenous sex steroid hormones from hSHBG binding sites and disrupt the androgen-to-estrogen balance. Whether xenoestrogen SHBG ligands could reach high enough concentrations in the blood to expose humans to any such effect merits further investigation.     

17beta-estradiol regulation of human growth hormone (hGH), insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) axis in hypoestrogenic, hypergonadotropic women

OBJECTIVE: Ovarian hormonal function may be as important contributing factor to hGH-IGF-I-IGFBP-3 axis as age. AIM: To examine plasma hGH, IGF-1 and IGFBP-3 levels in women with premature ovarian failure compared to healthy normal controls and postmenopausal ones. PATIENTS: Group A-15 women with premature ovarian failure (POF) (mean: age 38.9+/-5.2 years, FSH 101.4+/-29.0 IU/l; 17beta-estradiol 22.5+/-14.6 ng/l). Group B consisted of 15 menopausal women (mean: age 54.7+/-2.7 years; FSH 81.9+/-32.1 IU/l; 17beta-estradiol 17.1+/- 8.0 ng/l). Group C - controls - 15 normally menstruating women (mean: age 37.1+/-9.0 years; FSH 6.2+/-1.0 IU/l; 17beta-estradiol 144.8+/-117.1 ng/l). METHODS: Body mass and BMI were measured. Basic fasting plasma hGH, IGF-I, IGFBP-3, insulin, testosterone and LH as well as prolactin (PRL), FSH and estradiol were assessed by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney u-test, Spearman rang correlation coefficient, stepwise multiple regression. RESULTS: Mean serum IGF-I level was the lowest (p<0.005) in group B (172.0+/-54.6 microg/l) and the highest in group C (273.6+/-109.0 microg/l). The mean plasma IGF-I level in group A was similar (NS) (208.3+/-66.5 microg/l) to that found in group B and lower (p<0.02) compared with that in group C. The lowest (p<0.005) serum IGFBP-3 level was found in group B (3.1+/-0.7 microg/l) compared to group C (4.4+/-0.3 microg/l). The mean plasma IGFBP-3 level (3.1+/-1.0 microg/l) in group A was lower than in group C (p<0.005) but identical as in group B. No statistically significant differences between groups were observed in mean hGH levels. Women in group A and C were younger (p<0.001) than those in group B. The lowest mean estradiol level was found in groups A and B. The highest was in group C (p<0.001). Mean plasma LH and FSH levels were higher (p<0.001) in groups A and B vs group C. In group C there were links between IGF-I and age (r=-0.60; p=0.014) The IGF-I/age relation disappeared in the groups A and B (rA=-0.26; rB=0.10; NS). The same regards IGFBP-3/ age link (rA=-0.44, NS; rB=0,31;NS). Estradiol level was related to hGH levels in group C (r=-0.54; p<0.05). In none of groups hGH/IGF-1 as well as IGFBP-3/hGH relations were found. Prolactin accounted for 69% of the variance in IGF-I level in the group B (p=0.003) and for 24% in group A (NS). Testosterone accounted for 88% (p=0.004) of the variance in IGF-I level in group B and IGFBP-3 was responsible for 86% (p=0.038) of the variance in IGF-I level in group C. Again IGFBP-3 was responsible for 47% (p=0.023) in group A and for 49% (p=0.04) in group B of the hGH variance. CONCLUSIONS: 17b-estradiol may be as important contributor to insulin-like growth factor-I (IGF-I) plasma level as age in hypoestrogenic, hypogonadotropic women.     

Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.     

甲胺磷和17β－雌二醇对萼花臂尾轮虫实验种群动态的影响

对暴露于不同浓度的甲胺磷(0.01、0.1、1.0、10.0、100.0、1 000.0和10 000.0 μg·L-1)和17β-雌二醇(0.001、0.01、0.1、1.0、10.0、100.0、和1 000.0 μg·L-1)溶液中的萼花臂尾轮虫的种群动态进行了研究.结果表明:甲胺磷和17β-雌二醇对轮虫种群动态的影响存在差异.甲胺磷和17β-雌二醇对实验期间轮虫的平均种群增长率均有显著影响,但对轮虫种群中的混交雌体受精率的平均值无显著影响;甲胺磷对实验期间轮虫种群中的携卵雌体数/非携卵雌体数和混交雌体百分率的平均值均有显著影响,而17β-雌二醇则无显著影响;甲胺磷对实验期间轮虫的休眠卵总产量无显著影响,而17β-雌二醇的影响较显著.与对照相比,10.0～10 000.0 μg·L-1的甲胺磷和100.0 μg·L-1的17β-雌二醇均使轮虫的平均种群增长率显著提高;0.1～10 000.0和10.0 μg·L-1的甲胺磷分别显著地降低了实验期间轮虫种群中的携卵雌体数/非携卵雌体数和混交雌体百分率的平均值;1 000.0 μg·L-1的17β-雌二醇显著降低了轮虫的休眠卵总产量.在实验浓度范围内,轮虫平均种群增长率(Y,d-1)与甲胺磷浓度(X,μg·L-1)之间的关系为Y=-2×10-8X2 0.0002X 0.3474.种群增长率可用于监测和评价甲胺磷对轮虫种群动态的影响.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 &#119 l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 μl each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Effects of octylphenol and 17beta-estradiol on the gonads of guppies (Poecilia reticulata) exposed as adults via the water or as embryos via the mother

Endocrine disrupting alkylphenolic compounds have been found in the aquatic environment, and concern has arisen over the ability of these compounds to affect the reproductive system of fish. In this study, the effects of exposure to an environmentally relevant concentration of octylphenol or 17beta-estradiol on the gonad structure of fish were examined. Viviparous guppies (Poecilia reticulata) were exposed as adults via the water or as embryos via the mother to 26 microg/l octylphenol or 0.85 microg/l 17beta-estradiol (mean measured water concentrations). Histological examinations revealed effects of the exposures on the gonads of the fish exposed as adults. Indications of blocked spermatogonial mitosis were seen in the testis structure of adult males after exposure to octylphenol or 17beta-estradiol. The post-parturition ovaries of adult females exposed to 17beta-estradiol showed effects suggesting an inhibited yolk deposition. At the tested concentrations, exposure to octylphenol or 17beta-estradiol via the mother fish did not significantly affect the weight, length, gonopodium index or sex distribution of the offspring. However, histology revealed effects on the liver structure, suggesting some effect of maternal exposure to octylphenol or 17beta-estradiol. These findings indicate that although octylphenol and 17beta-estradiol affect the gonad structure of adult male and female guppies, these substances have no significant effects on the sexual differentiation of the embryos at the tested concentrations.     

Effects of selected xenoestrogens on liver peroxisomes, vitellogenin levels and spermatogenic cell proliferation in male zebrafish

Environmental estrogenic compounds or xenoestrogens can mimic natural estrogens and cause a variety of adverse effects on aquatic wildlife. The purpose of the present work was to investigate if xenoestrogens are able to cause proliferation of liver peroxisomes using zebrafish (Danio rerio) as a model. Adult male zebrafish were exposed for 15 days to 17beta-estradiol (E2) and the xenoestrogens dibutylphthalate (DBP), methoxychlor (MXC), 4-tert-octylphenol (OP) and 17alpha-ethynylestradiol (EE2). All five tested compounds caused significant proliferation of liver peroxisomes (p < 0.05) as indicated by increased peroxisomal surface and numerical densities and elevated activities of the peroxisomal beta-oxidation enzyme acyl-CoA oxidase (AOX). In the case of DBP, MXC and E2, positive significant correlations between peroxisomal density parameters and AOX were found. The treatments did not produce gross alterations in testis histology, but spermatogenic cell proliferation was disturbed in E2 and EE2-treated groups and vitellogenin levels increased significantly in fish exposed to MXC, OP, EE2 and E2 with respect to controls. Furthermore, a significant correlation between vitellogenin levels and AOX activity was found for MXC, OP and EE2 treatments, suggesting that for the latter xenoestrogens early estrogenic effects are associated with liver peroxisome proliferation. No such association occurred with typical peroxisome proliferators such as DBP.     

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.)

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.     

Development of a micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for rapid- and high-throughput analysis of 17beta-estradiol in water samples

A novel method of micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for the screening of 17beta-estradiol in water samples was proposed. It used the micro-plate magnetic separator designed by ourselves which can achieve the high-throughput analysis without the samples pre-treatment and the sensitive chemiluminescence system of AMPPD-ALP system. The method showed specific recognition of estrogen, without cross-reactions for three other major estrogenic compounds (17beta-estradiol (E2), estriol (E3), ethinyl (E2)) commonly found in water. The MMCLEIA was also especially suitable for the large-scale samples processing. The working range for 17beta-estradiol was 10-3000 pg/ml. The assay sensitivity was 5.4 pg/ml. Both intra- and inter-assay had relative standard deviation of less 15%. The effect of several physico-chemical parameters, such as the ratio of antibody versus antigen, incubation time and the concentration of detergent were studied. This method has been successfully applied to the preliminary detection of the sea-water. Compared with the chemiluminescence enzyme-linked immunosorbed assay, the correlation was good.     

Antidepressant-like effect of 17beta-estradiol: involvement of dopaminergic, serotonergic, and (or) sigma-1 receptor systems

17beta-estradiol has been reported to possess antidepressant-like activity in animal models of depression, although the mechanism for its effect is not well understood. The present study is an effort in this direction to explore the mechanism of the antidepressant-like effect of 17beta-estradiol in a mouse model(s) of behavioral depression (despair behavior). Despair behavior, expressed as helplessness to escape from a situation (immobility period), as in a forced swim test in which the animals are forced to swim for a total of 6 min, was recorded. The antiimmobility effects (antidepressant-like) of 17beta-estradiol were compared with those of standard drugs like venlafaxine (16 mg/kg, i.p.). 17beta-estradiol produced a U-shaped effect in decreasing the immobility period. It had no effect on locomotor activity of the animal. The antidepressant-like effect was comparable to that of venlafaxine (16 mg/kg, i.p.). 17beta-estradiol also exhibited a similar profile of antidepressant action in the tail suspension test. When coadministered with other antidepressant drugs, 17beta-estradiol (5 microg/kg, i.p.) potentiated the antiimmobility effect of subeffective doses of fluoxetine (5 mg/kg, i.p.), venlafaxine (2 mg/kg, i.p.), or bupropion (10 mg/kg, i.p.), but not of desipramine (5 mg/kg, i.p.) or tranylcypromine (2 mg/kg, i.p.), in the forced swim test. The reduction in the immobility period elicited by 17beta-estradiol (20 microg/kg, i.p.) was reversed by haloperidol (0.5 mg/kg, i.p.; a D(2) dopamine receptor antagonist), SCH 23390 (0.5 mg/kg, i.p.; a D(1) dopamine receptor antagonist), and sulpiride (5 mg/kg, i.p.; a specific dopamine D(2) receptor antagonist). In mice pretreated with (+)-pentazocine (2.5 mg/kg, i.p.; a high-affinity sigma-1 receptor agonist), 17beta-estradiol (5 microg/kg, i.p.) produced a synergistic effect. In contrast, pretreatment with progesterone (10 mg/kg, s.c.; a sigma-1 receptor antagonist neurosteroid), rimcazole (5 mg/kg, i.p.; another sigma-1 receptor antagonist), or BD 1047 (1 mg/kg, i.p.; a novel sigma-1 receptor antagonist) reversed the antiimmobility effects of 17beta-estradiol (20 microg/kg, i.p.). Similarly, in mice pretreated with a subthreshold dose of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, a 5-HT1A serotonin receptor agonist), 17beta-estradiol (5 microg/kg, i.p.) produced an antidepressant-like effect. These findings demonstrate that 17beta-estradiol exerted an antidepressant-like effect preferentially through the modulation of dopaminergic and serotonergic receptors. This action may also involve the participation of sigma-1 receptors.     

Differential effects of natural and environmental estrogens on endothelin synthesis in bovine oviduct cells

Endothelin-1 (ET-1), a vasoconstrictor and mitogenic peptide that plays an important role within the endocrine/reproductive system, is synthesized by oviduct cells and regulates tubal contractility. Because 17beta-estradiol (estradiol) regulates oviduct function by influencing the synthesis of autocrine/paracrine factors, estradiol may also regulate ET-1 synthesis. Furthermore, environmental estrogens (EEs; phytoestrogens and xenoestrogens), which structurally resemble estradiol and possess estrogenic activity, may mimic the effects of estradiol on ET-1 synthesis and may influence the reproductive system. Using cultures of bovine oviduct cells (epithelial cells:fibroblasts, 1:1), we investigated and compared the modulatory effects of estradiol, phytoestrogens, and xenoestrogens on ET-1 synthesis and determined whether these effects were estrogen receptor (ER) mediated. A quantitative ELISA for ET-1 in the culture medium revealed that 17beta-estradiol inhibits ET-1 synthesis in a concentration-dependent manner (4-400 nmol/L). In contrast to estradiol, ET-1 synthesis was induced in cell cultures treated with xenoestrogens in the following order of potency (0.1 micromol/L): 4-hydroxy-trichlorobiphenyl > 4-hydroxy-dichlorobiphenyl > trichlorobiphenyl. The stimulatory effects of xenoestrogens on ET-1 production were mimicked by the phytoestrogens biochanin-A and genistein but not by formononetin, equol, and daidzein. The oviduct cells expressed both ERs (alpha and beta), but the modulatory effects of estradiol, but not EEs, on ET-1 synthesis were blocked by ICI-182 780 (1 microM), a pure ER antagonist. Our results provide evidence that estradiol inhibits ET-1 synthesis in oviduct cells via an ER-dependent mechanism, whereas, EEs induce ET-1 synthesis via an ER-independent mechanism. The contrasting effects of EEs on ET-1 synthesis suggests that EEs may act as endocrine modulators/disruptors and may have deleterious effects on the reproductive system by adversely influencing the biology and physiology of the oviduct.     

Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

Effects of 17beta-estradiol and flutamide on splenic macrophages and splenocytes after trauma-hemorrhage

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.