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To investigate the degradation efficiency of conditioners and commercial microbial agents on estrogens (E1, 17α-E2, 17β-E2, E3, EE2, and DES) in the composting process of dairy manure, seven different treatments (RHB-BF, OSP-BF, SD-BF, MR-BF, MR-FS, MR-EM, and MR-CK) under forced ventilation conditions were composted and monitored regularly for 30 days. The results indicated that the removal rates of estrogens in seven treatments ranged from 95.35% to 99.63%, meanwhile the degradation effect of the composting process on 17β-Estradiol equivalent (EEQ) was evaluated, and the removal rate of ΣEEQ ranged from 96.42% to 99.72%. With the combined addition of rice husk biochar (RHB) or oyster shell powder (OSP) and bio-bacterial fertilizer starter cultures (BF), namely RHB-BF and OSP-BF obviously promoted the rapid degradation of estrogens. 17β-E2 was completely degraded on the fifth day of composting in OSP-BF. Microbial agents have some promotional effect and enhances the microbial degradation of synthetic estrogen (EE2, DES). According to the results of RDA, pH and EC were the main environmental factors affecting on the composition and succession of estrogen-related degrading bacteria in composting system. As predominant estrogens-degrading genera, Acinetobacter, Bacillus, and Pseudomonas effected obviously on the change of estrogens contents. The research results provide a practical reference for effective composting of dairy manure to enhancing estrogens removal and decreasing ecological risk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

17α-ethinylestradiol (EE2) is a synthetic estrogen that can cause harmful effects on animals, such as male feminization and infertility. However, the impact of the EE2 contamination on microbial communities and the potential role of bacterial strains as bioremediation agents are underexplored. The aim of this work was to evaluate the impact of EE2 on the microbial community dynamics of aerated submerged fixed-film reactors (ASFFR) simulating a polishing step downstream of a secondary sewage treatment. For this purpose, the reactors were fed with a synthetic medium with low COD content (around 50 mg l -1 ), supplemented (reactor H) or not (reactor C) with 1 μg l -1 of EE2. Sludge samples were periodically collected during the bioreactors operation to assess the bacterial profile over time by 16S rRNA gene amplicon sequencing or by bacterial isolation using culture-dependent approach. The results revealed that the most abundant phyla in both reactors were Proteobacteria and Bacteroidetes. At genus level, Chitinophagaceae, Nitrosomonas and Bdellovibrio predominated. Significant effects caused by EE2 treatment and bioreactors operating time were observed by non-metric multidimensional scaling. Therefore, even at low concentrations as 1 μg l -1 , EE2 is capable of influencing the bioreactor microbiome. Culture-dependent methods showed that six bacterial isolates, closely related to Pseudomonas and Acinetobacter genera, could grow on EE2 as the sole carbon source under aerobic conditions. These organisms may potentially be used for the assembly of an EE2-degrading bacterial consortium and further exploited for bioremediation applications, including tertiary sewage treatment to remove hormone-related compounds not metabolized in secondary depuration stages., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Very little is currently known regarding the effects of estrogenic endocrine disrupting chemicals on embryonic and larval development in molluscs, nor the potential effects of parental (F 0 ) exposure on resultant F 1 offspring. In this study, we assessed the embryotoxic impacts of exposure to environmentally relevant concentrations of the synthetic estrogen, 17α-ethinylestradiol (EE2), to male and female parents (50 ng/L) and their offspring (5 and 50 ng/L) in the native Australian Sydney rock oyster, Saccostrea glomerata. There were no detectable effects of parental exposure on fertilisation success, proportions of early larval (F 1 ) morphs and unfertilised eggs. Offspring impacts were evidenced in terms of developmental delays, with decreased percentages of D-veligers retained by 45 μm mesh, along with a reduction of swimming capabilities of larvae at 2 days post-fertilisation (dpf) when both parents had been exposed to 50 ng/L EE2. Although no significant parental effects were found on the survival of F 1 larvae at 9 dpf, retardation of shell growth was observed on F 1 larvae in treatments where both parents had been exposed to 50 ng/L EE2. Subsequent larval exposure from 2 to 9 dpf caused declines in survival and reduction of shell length in F 1 larvae at both 5 and 50 ng/L EE2 across all parental exposure treatments. Collectively, parental EE2 imparts effects on offspring in terms of retardation of larval development, and subsequent offspring exposure to EE2 further exacerbates impacts to development. Future research should aim to understand the potential mechanisms of EE2 induced toxicity and its transmission resulting in altered phenotypes of the F 1 generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cyanobacteria blooms are increasing globally, with further increases predicted in association with climate change. Recently, some cyanobacteria species have been identified as a source of estrogenic effects in aquatic animals. To explore possible estrogenic effects of Microcystis aeruginosa (an often-dominant cyanobacteria species) on zooplankton, we examined effects of cyanobacteria exudates (MaE, 2 × 10 4 and 4 × 10 5 cells/ml) on reproduction in Daphnia magna. We analyzed physiological, biochemical and molecular characteristics of exposed Daphnia via both chronic and acute exposures. MaE at both low and high cell density enhanced egg number (15.4% and 23.3%, respectively) and reproduction (37.7% and 52.4%, respectively) in D. magna similar to 10 μg/L estradiol exposure. In addition, both MaE of low and high cell densities increased population growth rate (15.8% and 19.6%, respectively) and reproductive potential (60% and 83%, respectively) of D. magna. These exudates promoted D. magna reproduction by stimulating 17β-hydroxysteroid-dehydrogenase (17β-HSD) activity and production of ecdysone and juvenile hormone, and by enhancing vitellogenin biosynthesis via up-regulating expression of Vtg1 and Vtg2. However, increased expression (6.6 times higher than controls) of a detoxification gene (CYP360A8) indicated that MaE might also induce toxicity in D. magna. Reproductive interference of zooplankton by blooming cyanobacteria might negatively affect foodwebs because MaE-induced zooplankton population increase would enhance grazing and reduce abundance of edible algae, thereby adding to the list of known disruptive properties of cyanobacterial blooms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Natural steroid estrogens (NSEs), including free estrogens (FEs) and conjugated estrogens (CEs), are of emerging concern globally among public and scientific community due to their recognized adverse effects on human and wildlife endocrine systems in recent years. In this review, the properties, occurrence, sorption process, and transformation pathways of NSEs are clarified in the environment. The work comprehensively summarizes the occurrence of both free and conjugated estrogens in different natural and built environments (e.g., river, WWTPs, CAFOs, soil, and sediment). The sorption process of NSEs can be impacted by organic compounds, colloids, composition of clay minerals, specific surface area (SSA), cation exchange capacity (CEC), and pH value. The degradation and transformation of free and conjugated estrogens in the environment primarily involves oxidation, reduction, deconjugation, and esterification reactions. Elaboration about the major, subordinate, and minor transformation pathways of both biotic and abiotic processes among NSEs is highlighted. The moiety types and binding sites also would affect deconjugation degree and preferential transformation pathways of CEs. Notably, some intermediate products of NSEs still remain estrogenic potency during transformation process; the elimination of total estrogenic activity needs to be addressed in further studies. The in-depth researches regarding the behavior of both free and conjugated estrogens are further required to tackle their contamination problem in the ecosystem. Graphical abstract ᅟ.

The present endocrine disrupting chemicals (EDCs) in wastewater effluents due to incomplete removal during the treatment processes may cause potential ecological and human health risks. This study evaluated the removal and uptake of seven EDCs spiked in two types of wastewater effluent (i.e., ultrafiltration and ozonation) and effluent cultivated with the freshwater green alga Nannochloris sp. In ultrafiltration effluent cultivated with Nannochloris sp. for 7 days, the removal rate of 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and salicylic acid (SAL) was 60%; but Nannochloris sp. did not promote the removal of other EDCs studied. The algal-mediated removal of E2, EE2, and SAL was attributed to photodegradation and biodegradation. Triclosan (TCS) underwent rapid photodegradation regardless of adding algae in the effluent with 63%-100% removal within 7 days. Triclosan was also found associated with algal cells immediately after adding algae, and thus the primary mechanisms involved were photodegradation and bioremoval (i.e., bioadsorption and bioaccumulation). After algal cultivation, TCS still has a bioaccumulation potential to pose high risks within the food web and the endocrine disrupting properties of the residual estrogens in the effluent are not eliminated. Algal cultivation can be exploited to treat wastewater effluents but the removal efficiencies of EDCs highly depend on chemical types., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Halogenated estrogens are thought to be moderately potent endocrine-disrupting compounds that are formed during chlorine-based wastewater disinfection processes and may represent a significant fraction of the total amount of estrogen delivered from wastewater treatment plants to receiving waters. Yet we lack key information about the photochemical degradation of halogenated estrogens, a process that has important implications for UV-based wastewater treatment and environmental fate modeling. To better understand halogenated estrogen degradation in aquatic environments, we studied the direct photolysis of 17β-estradiol (E2), 2-bromo-17β-estradiol (monoBrE2), 2,4-dibromo-17β-estradiol (diBrE2), and 2,4-dichloro-17β-estradiol (diClE2) as well as the indirect photolysis of diBrE2 under natural solar irradiance. We found that direct photolysis rate constants increased with halogenation as pKa values decreased and molar absorptivity spectra shifted toward higher wavelengths. Compared to E2, quantum yields were threefold larger for monoBrE2, but 15-32% smaller for the dihalogenated forms. The rate of diBrE2 (pKa ∼ 7.5) photolysis was strongly influenced by pH. At pH 7, diBrE2 degraded on minute time scales due to the large red-shifted molar absorptivity values and greater quantum yields of the phenolate form. Degradation rates were only slightly different in the presence of Suwannee River Humic Acid (5 mg L-1), and quenching experiments pointed to excited triplet state dissolved organic matter (3DOM*) as the dominant reactive intermediate responsible for the indirect photolysis of diBrE2. Overall, our data suggest that halogenated estrogens are particularly susceptible to photochemical degradation at environmentally relevant pH values.

Hormone active agents constitute a dangerous class of pollutants. Among them, those agents that mimic the action of estrogens on target cells and are part of the group of endocrine-disruptor compounds (EDCs) are termed estrogenic EDCs, the main focus of this review. Exposure to these compounds causes a number of negative effects, including breast cancer, infertility and animal hermaphroditism. However, especially in underdeveloped countries, limited efforts have been made to warn people about this serious issue, explain the methods of minimizing exposure, and develop feasible and efficient mitigation strategies at different levels and in various environments. For instance, the use of bioremediation processes capable of transforming EDCs into environmentally friendly compounds has been little explored. A wide diversity of estrogen-degrading microorganisms could be used to develop such technologies, which include bioremediation processes for EDCs that could be implemented in biological filters for the post-treatment of wastewater effluent. This review describes problems associated with EDCs, primarily estrogenic EDCs, including exposure as well as the present status of understanding and the effects of natural and synthetic hormones and estrogenic EDCs on living organisms. We also describe potential biotechnological strategies for EDC biodegradation, and suggest novel treatment approaches for minimizing the persistence of EDCs in the environment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study deals with the use of low-cost plant-derived materials, namely a biochar, spent coffee grounds, spent tea leaves, and a compost humic acid, for the adsorptive removal from water of two estrogens, 4-tert-octylphenol (OP) and 17-β-estradiol (E2), and two pesticides, carbaryl and fenuron, each spiked at a concentration of 1 mg L -1 . Kinetics and adsorption isotherms have been performed using a batch equilibrium method to measure the sorption capacities of the adsorbents towards the four molecules. Adsorption constants were calculated using the linear, Freundlich, and Langmuir models. Kinetics data obtained evidenced a rapid adsorption of each compound onto both biochar and coffee grounds with the attainment of a steady-state equilibrium in less than 4 h. Significant differences among the adsorbents and the compounds were found regarding the model and the extent of adsorption. In general, the estrogens were adsorbed more quickly and in greater amounts than the less hydrophobic pesticides, following the order: OP > E2 > carbaryl > fenuron. The ranges of Freundlich constants obtained for OP, E2, carbaryl, and fenuron onto the sorbents were 5049-2253, 3385-206, 2491-79, and 822-24 L kg -1 , respectively. The maximum values of constants were obtained for biochar, except for OP that was more adsorbed by spent coffee grounds. Adsorption kinetic data followed a pseudo-second-order model, thus indicating the occurrence of chemical interactions between the compounds and the substrates. The remarkable sorption capacities of all adsorbents towards the four molecules suggest the valuable exploitation of these materials for decontamination purposes, such as the treatment of wastewater before a feasible recycle in soil.

The present work describes the development of a novel fully automated online solid phase extraction (SPE) coupled with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using negative electrospray ionisation (ESI) for the simultaneous determination of six estrogens and six glucocorticoids in water. Filtered water samples (5mL) were preconcentrated on a HyperSep™ Retain PEP SPE cartridge, eluted in back-flush mode, and separated on an LC column before analysis by tandem mass spectrometry. The total analysis time for each sample was 17min. Different experimental parameters such as the type of online SPE cartridge, loading flow rate, and composition of methanol in the loading phase were optimised. The intra-day repeatability of method ranged from 1.48 to 9.68% for all analytes, and the inter-day reproducibility ranged from 2.03 to 8.63% for all analytes, except for dexamethasone at 11.95%. These were calculated based on the peak area responses of the targeted analytes spiked at 50ng/L in ultrapure water. The method also showed good linearity from 1 to 100ng/L, with the limits of detection (LODs) ranging from 0.16 to 2.14ng/L. The proposed method was applied to the analysis of municipal wastewater. This fully automated online SPE extraction coupled with LC-MS/MS method is effective and reliable to measure estrogens and glucocorticoids simultaneously due to its high throughput, relatively low solvent consumption, reusability of the online SPE cartridge, and reduction of manual labor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Estrogens are known to be potent endocrine disrupting chemicals that are commonly found in wastewater effluents at ng L(-1) levels. Yet, we know very little about the distribution and fate of estrogens in coastal oceans that receive wastewater inputs. This study measured a wide range of steroidal estrogens in sewage-impacted seawater using ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) together with the method of standard addition. In Massachusetts Bay, we find conjugated, free, and halogenated estrogens at concentrations that are consistent with dilution at sites close to the sewage source. At a site 6 miles down current of the sewage source, we observe estrone (E1) concentrations (520 ± 180 pg L(-1)) that are nearly double the nearfield concentrations (320 ± 60 pg L(-1)) despite 9-fold dilution of carbamazepine, which was used as a conservative sewage tracer. Our results suggest that background E1 concentrations in Massachusetts Bay (∼270 ± 50 pg L(-1)) are derived largely from sources unrelated to wastewater effluent such as marine vertebrates.

Pregnant eelpout were exposed via the water to known endocrine disrupting compounds (EDCs) to clarify if EDCs could be causing the increased eelpout fry malformation frequencies observed in coastal areas receiving high anthropogenic input. The presence of a teratogenic window for estrogen-induced malformations was also investigated by starting the exposure at different times during eelpout pregnancy. Both 17α-ethinylestradiol (EE2) (17.8 ng/L) and pyrene (0.5 μg/L) significantly increased fry malformation frequency whereas 4-t-octylphenol (4-t-OP) up to 14.3 μg/L did not. Vitellogenin was significantly induced by EE2 (5.7 and 17.8 ng/L) but not by 4-t-OP and pyrene. A critical period for estrogen-induced fry malformations was identified and closed between 14 and 22 days post fertilization (dpf). Exposure to 17β-estradiol (E2) between 0 and 14 dpf caused severe malformations and severity increased the closer exposure start was to fertilization, whereas malformations were absent by exposure starting later than 14 dpf. Data on ovarian fluid volume and larval length supported the suggested teratogenic window. Larval mortality also increased when exposure started right after fertilization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity and the effects on the steroidogenesis. In the present study, we aim to explore the effects of BPA on epigenetic modification in rare minnow Gobiocypris rarus. We have detected the global and cyp19a1a gene specific DNA methylation in gonads of adult G. rarus under BPA exposure. The global DNA methylation level was significantly increased in testis of the male fish exposed to BPA for 7 days, and it was significantly increased in the ovary following 35-days exposure. DNA methyltransferases (DNMTs) catalyze the transfer of a methyl moiety from S-adenosyl-l-methionine to the cytosine of a CpG dinucleotide. The alteration of the detected dnmts mRNA expression could affect the global DNA methylation levels following 15μg/L BPA exposure. Cytochrome P450 aromatase (CYP19A1A), is responsible for the conversion of androgens into estrogens, which plays a vital role in estrogen synthesis in gonads. In the present study, the methylation level of ovarian cyp19a1a gene was significantly suppressed and stimulated by 7- and 35-day BPA exposure, respectively. There was a significant negative correlation between cyp19a1a mRNA expression and methylation levels of the four CpGs at the 5' flanking region in the ovary of adult G. rarus following BPA exposure. So we hypothesize that there are some association between the reproductive toxicity of BPA and the global DNA methylation under BPA exposure. And the alteration of cyp19a1a expression in female G. rarus by BPA might attribute to the change of its DNA methylation status., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A method has been developed for the simultaneous determination of six estrogens: (estriol, 17-beta-estradiol, 17-alpha-estradiol, estrone, ethinyloestradiol, diethylstilbestrol) in water environment by solid phase extraction and ultra performance liquid chromatography couple with triple quadrupole mass spectrometry. After enriched and purified by an HLB solid phase extraction column, the six estrogens were separated by a BEH C18 column and then determined by MS/MS with multiple reaction monitoring (MRM) mode. The quantification was achieved by internal standard method, with estriol-D3, estradiol-D2 and diethylstilbestrol-D8 as the internal standards. Good linear relationships were obtained in the mass concentration range of 1.0-100 microg/L with high correlation coefficients over 0. 998 2. The limits of detection and quantification for the six estrogens were 0.27-0.45 ng/L and 1.08-1.78 ng/L, respectively. The recoveries were in the range of 68.3%-97.4% at the three spiked levels and with the relative standard deviations below 15%. The method shows high sensitivity, good precision, extensive analytical range and high analytical speed. It was successfully applied to the simultaneous detection of the six estrogens in four different water bodies including surface water, waste water, source water of drinking and domestic water.

Objective: To investigate Nova Scotia (NS) pharmacists' knowledge and beliefs regarding the use of bioidentical hormones (BHs) for the management of menopause related symptoms., Methods: Using Dillman's tailored design methodology, an invitation to complete the web-based questionnaire was emailed to pharmacists in NS as part of the Dalhousie College of Pharmacy Continuing Pharmacy Education Department's (CPE) weekly email update. Data was analyzed using descriptive statistics., Results: Of approximately 1300 e-mails sent, 113 pharmacists completed the questionnaire (response rate 8.7%). The majority of respondents (94%) knew that BHs were not free from adverse drug reactions. More than 50% were aware that conjugated equine estrogens and medroxyprogesterone acetate were not examples of BHs. For seven of eleven knowledge questions, 33-45% indicated that they did not know the answer. When asked about their beliefs regarding BHs, many believed that BHs were similar in efficacy (49%) or more effective (21%) than conventional hormone therapy (CHT) for vasomotor symptoms. Most respondents also believed that both BHs and CHT had similar safety profiles. Additionally, responding pharmacists indicated that more education would be helpful, especially in the area of safety and efficacy of BHTs compared to CHT., Conclusions: NS pharmacists knew BHs were not free of adverse effects, however knowledge was lacking in other areas. This may reflect the level of coverage of this topic in pharmacy school curriculums and in the pharmacy literature. Results indicate a need for additional education of NS pharmacists with respect to BHs, which could be accomplished through modification of undergraduate pharmacy programs and supplementary CPE.

Background: The overwhelming majority of patients with stress cardiomyopathy (SC) are postmenopausal women, suggesting an important pathophysiologic role of the female sex hormones. Preliminary data suggest that myocardial stunning might be provoked by estrogen deficiency., Hypothesis: We hypothesized that, compared with age- and gender-matched patients with myocardial infarction (MI) or patients with normal coronary arteries, patients with SC would exhibit altered levels of sex hormones. Furthermore, we aimed to describe the clinical course and the pattern of sex hormones of the SC patients during long-term follow-up., Methods: Blood samples obtained on hospital admission were analyzed for estradiol (E2), progesterone (P), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in women with SC (n = 17), age-matched women with acute MI (n = 16), and women with normal coronary arteries (n = 15). Six years after the initial event, SC patients underwent a clinical and echocardiographic follow-up and reassessment of sex hormones., Results: Estrogen concentrations at hospital admission were significantly higher in the SC group compared with the MI and the control groups, with no difference in P, FSH, and LH concentrations. Follow-up E2 after 6 years in SC patients was lower than during the acute SC episode. Follow-up P in these patients was lower than P in the MI and control groups during the acute event, with a similar trend for E2. After a median follow-up of 6.4 years, 1 sudden cardiac death occurred and 2 patients suffered from SC recurrence., Conclusions: During the acute event, E2 concentrations are elevated in postmenopausal SC patients compared with women with acute MI or with normal coronary arteries. The higher E2 concentrations might have exerted atheroprotective effects and thus diverted the stress response to SC rather than MI. Recurrence and/or sudden cardiac death remains a potential risk of SC., (© 2012 Wiley Periodicals, Inc.)

A passive sampler (the polar organic chemical integrative sampler; POCIS) was assessed for its ability to sample natural estrogens (17β-estradiol, E2; estrone, E1 and estriol, E3) and the synthetic estrogen (17α-ethynylestradiol, EE2) in the outlet of a sewage treatment works over several weeks. The performance of the POCIS was investigated and optimised in the laboratory before field deployment with high recoveries (66-99%) were achieved for all estrogens. Moreover, it was shown that POCIS does not exhibit any preferential selectivity towards any of the target compounds. The sampling rates of E1, E2 and E3 were 0.018 ± 0.009, 0.025 ± 0.014 and 0.033 ± 0.019 L d(-1), respectively. Following field deployments of 28 days in the discharge of a sewage works, POCIS was shown to enhance the sensitivity of estrogen detection, especially for E3, and provide time-weighted average (TWA) concentrations of E1, E2 and E3, ranging from undetectable to 12 ng L(-1) upstream of the outflow of a sewage treatment works, 13 to 91 ng L(-1) at the outflow and 8 to 39 ng L(-1) downstream of the outflow. This revealed that E1, E2 and E3 are not completely removed during sewage treatment, with concentrations most likely being maintained by contributions from conjugated estrogen analogues. Grab water samples showed considerable variation in the concentrations of estrogens over a longer period (6 months). The results confirm that POCIS is an effective and non-discriminatory method for the detection of low concentrations of estrogens in the aquatic environment.

Colchicine (CAS 64-86-8) is considered to have a hepatoprotective effect and play a role in biliary excretion. 17alpha-Ethynylestradiol (EE) (5 mg/kg, subcutaneously, daily, for 5 days) causes intrahepatic cholestasis by reducing both the influx and efflux of bile acid in hepatocytes, resulting in a decrease in bile flow. The objective of this study was to evaluate whether colchicine has any effect on EE-induced cholestasis. The effects of colchicine treatment on EE-induced cholestasis in rats for 5 consecutive days were evaluated. The serum components and enzymatic activity were assayed. In addition, the bile flow and biliary excretion were determined. Furthermore, western blot analysis was used to measure the expression of farnesoid X receptor (FXR), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and cholesterol 7alpha-hydroxylase (CYP7A1). Colchicine not only significantly inhibited the elevation of cholestasis-related serum components and enzyme activity but also significantly attenuated the decrease of the bile flow and biliary excretion. Colchicine also remarkably increased the hepatic expression of FXR, BSEP and MRP2, but decreased that of CYP7A1. Our data indicates that colchicine treatment attenuated EE-induced cholestasis in rats, most likely by promoting bile flow and biliary excretion, and reduced the synthesis of bile acids.

Stroke is the leading cause of disability in the United States and affects 15 million people worldwide. Studies performed in various parts of the world have found differences between sexes in stroke incidence, prevalence, mortality, and outcomes. Although men are at higher risk of stroke for most age groups below age 85 years, after this age the incidence reverses dramatically, with women being much more at risk. Furthermore, recent studies suggest that women have worse recovery than men post-stroke. Many aspects of recovery may influence this outcome, including sex-specific comorbidities, aggressiveness of acute treatment, prevention therapies, and varying degrees of social support and rates of depression. It is important to further define and investigate sex differences in stroke incidence, care, treatment, and outcomes to improve functional recovery in women.

There are many reports documenting the adverse effects, such as feminization of fish, of estrogen hormones in the environment. One of the major sources of these compounds is from municipal wastewater effluents. The biological processes at municipal wastewater treatment plants cannot completely remove these compounds. This paper discusses the use of ultrasound to destroy estrogen compounds in water. The study examines the effect of ultrasound power density and power intensity on the destruction of various estrogen compounds which include: 17alpha-estradiol, 17beta-estradiol, estrone, estriol, equilin, 17alpha-dihydroequilin, 17alpha-ethinyl estradiol and norgestrel. These tests were conducted in single component batch and flow through reactors using 0.6, 2 and 4kW ultrasound sources. The sonolysis process produced 80-90% destruction of individual estrogens at initial concentration of 10microg/L within 40-60min of contact time. First order rate constants for the individual compounds under different conditions are presented. The estrogen degradation rates increase with increase in power intensity. However, the energy efficiency of the reactor was higher at lower power density. The 4kW ultrasound reactor was more energy efficient compared to the 0.6 and 2kW sonicators.

In order to protect consumers from ultraviolet (UV) radiation and enhance light stability of the product, three to eight UV filters are usually added to consumer sunscreen products. High lipophilicity of the UV filters has been shown to cause bioaccumulation in fish and humans, leading to environmental levels of UV filters that are similar to those of PCBs and DDT. In this paper, estrogen-regulated pS2 gene transcription in the human mammary tumor cell line MCF-7 was used as a measure of estrogenicity of four individual UV filters. Since humans are exposed to more than one UV filter at a time, an equipotent binary mixture of 2-hydroxy-4-methoxy-benzophenone (BP-3) and its metabolite 2,4-dihydroxy benzophenone (BP-1), as well as an equipotent multi-component mixture of BP-1, BP-3, octyl methoxy cinnamate (OMC) and 3-(4-methylbenzylidene) camphor (4-MBC), were also evaluated for their ability to induce pS2 gene transcription in order to examine additivity. An estrogen receptor-mediated mechanism of action was expected for all UV filters. Therefore, our null-hypothesis was that combined estrogenic responses, measured as increased pS2 gene transcription in MCF-7 cells after exposure to mixtures of UV filters, are additive, according to a concentration-addition model. Not all UV filters produced a full concentration-response curve within the concentration range tested (100 nM-1 microM). Therefore, instead of using EC50 values for comparison, the concentration at which each compound caused a 50% increase of basal pS2 gene transcription was defined as the C50 value for that compound and used to calculate relative potencies. For comparison, the EC50 value of a compound is the concentration at which the compound elicits an effect that is 50% of its maximal effect. Individual UV filters increased pS2 gene transcription concentration-dependently with C50 values of 0.12 microM, 0.5 microM, 1.9 microM, and 1.0 microM for BP-1, BP-3, 4-MBC and OMC, respectively. Estradiol (E2) had a C50 value of 4.8 pM. Experiments with equipotent mixtures all supported our null hypothesis that mixtures of UV filters act additively to activate the estrogen receptor (ER). In view of our results and observed plasma levels it cannot be excluded that daily exposure to sunscreen formulations may have estrogenic effects in humans.

Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.

Objective: Estrogen or combined hormone (estrogen-progestin) therapy is highly efficacious for managing the signs and symptoms of urogenital atrophy. A low, effective estrogen dose may enhance patient acceptance and reduce side effects., Methods: In this randomized, double-blind, multicenter clinical trial, 71 healthy postmenopausal women with vaginal atrophy (Vaginal Maturation Index < or =55) received either low-dose synthetic conjugated estrogens, A tablets (Cenestin) (SCE-A), 0.3 mg once daily, or placebo for 16 weeks., Results: Treatment with SCE-A for 16 weeks resulted in a highly significant (P<0.0001) mean increase of 17.7 in the Vaginal Maturation Index compared to a mean increase of 4.1 with placebo treatment. A significant estrogenic improvement was detected as early as 4 weeks (mean increase 14.6). Superficial cells were significantly increased from 2.1% at baseline to 15.9% at week 16 with SCE-A, and parabasal cells were significantly reduced from 23.0% at baseline to 1.6% at week 16 (P<0.01 between treatments for both). Vaginal pH was significantly decreased from 6.2 at week -2 to 5.2 at week 16 with SCE-A compared to placebo (P<0.0001). There were no significant differences between treatment groups in the incidence of treatment-emergent side effects or other measures of safety, except for urinary tract infection, which occurred more frequently in the placebo group., Conclusions: These results confirm the relatively rapid estrogenic effect and safety of a low-dose (0.3 mg/day) of slow-release SCE-A (Cenestin) in the treatment of vaginal atrophy in postmenopausal women.

Objectives: To investigate the cognitive effects of high-dose oral estrogen alone or in combination with oral methyltestosterone in postmenopausal women., Methods: Participants were tested with a randomized, double-blind design on the Identical Pictures, Cube Comparisons, Building Memory and Shape Memory tasks before and after 4 months of hormone treatment., Results: Women receiving estrogen and methyltestosterone maintained a steady level of performance on the Building Memory task, whereas those receiving estrogen alone showed a decrease in performance., Conclusions: These results indicate that the addition of testosterone to high-dose estrogen replacement exerts a protective effect on memory performance in postmenopausal women., (Copyright 2002 S. Karger AG, Basel)

Objective: To estimate incidence of endometrial hyperplasia, vaginal bleeding, and menopausal symptoms in women who changed from standard monthly cyclic hormone replacement therapy (HRT) to half-strength estrogen opposed by medroxyprogesterone acetate (MPA) at 6-month intervals., Methods: We identified 138 women aged 55-75 years who had regularly used HRT at a standard dosage (equivalent to 0.625 mg conjugated estrogen) opposed by cyclic monthly MPA. Each subject's HRT regimen was changed to 0.3 mg/day esterified estrogens (Estratab; Solvay Pharmaceuticals, Marietta, GA) combined with 14-day courses of MPA, 10 mg/day, every 6 months. Endometrial biopsy was repeated after 1 year of the new regimen. Any vaginal bleeding was reported in each patient's daily diary. Menopause symptoms were evaluated using the Greene Menopause Symptom Index., Results: Among 125 women who had biopsy after 1 year of the new regimen, endometrial hyperplasia was found in two (1.6%, 95% confidence interval 0.3%, 6.2%). Of the 125 women, 44% had scheduled bleeding, and 9.4% had unscheduled bleeding. Relative to baseline vasomotor score (range 0-6), an increase of at least 2 U was reported by 20% of subjects at 6 months and by 17% of subjects at 12 months., Conclusion: Most women aged at least 55 years can safely switch their HRT regimen from standard dosage HRT to low-dosage estrogen opposed by MPA at 6-month intervals. Moreover, this new HRT regimen causes little vaginal bleeding while maintaining adequate control of menopausal symptoms.

Objective: To determine the relationships among bone mineral density changes, bone marker changes, and plasma estrogens in postmenopausal women receiving estrogen replacement therapy., Design: A total of 406 postmenopausal women received 1,000 mg calcium and continuous esterified estrogens (0.3 mg, 0.625 mg, or 1.25 mg) or placebo daily for up to 24 months. Bone mineral density and bone marker measurements were determined at 6-month intervals; plasma estrogens were measured in a subset after 12, 18, and 24 months., Results: Esterified estrogens produced significant increases in bone mineral density (lumbar spine, hip) compared with baseline and placebo at 6, 12, 18, and 24 months. Bone markers decreased from baseline with all esterified estrogen doses relative to placebo. Bone marker changes at 6 months correlated negatively with bone mineral density changes at 24 months (correlation coefficient range = -0.122 to -0.439). The strongest correlation was noted for spine bone mineral density changes and serum osteocalcin. Mean plasma estrogen levels increased with esterified estrogen dose, and bone mineral density changes correlated positively with plasma estrogen levels. Positive bone mineral density changes were noted in treatment groups with plasma estradiol levels at and above 25 pg/mL., Conclusions: Esterified estrogens, at doses from 0.3 mg to 1.25 mg/day, unopposed by progestin, increase bone mineral density of the spine and hip in postmenopausal women. These bone mineral density changes correlated significantly with bone marker changes at 6 months and with plasma estrogens at 12, 18, or 24 months. Data variability minimizes the predictive value of the bone marker changes in monitoring individual therapy.

We evaluated the acceptability of a systematic estrogen replacement therapy (ERT) substitution program in a large U.S. health maintenance organization (HMO). Prescriptions for 14,601 enrollees were converted from Premarin tablets (PR) to Estratab tablets (ES). At the end of 6 months, 93.5% of the women continued to use ES, and 6.5% reverted to use of PR. We report the results of a telephone survey that included women randomly selected from three groups who participated in the substitution program. The groups were women whose prescriptions were converted from PR to ES (n = 253), women whose prescriptions were converted from PR to ES and back to PR (n = 250), and women who continued to use PR without converting to ES (n = 251). Two thirds of women who did not revert to use of PR were still using ES 3 years after conversion of their prescriptions. Women whose prescriptions were converted but who returned to use of PR cited both return of menopausal symptoms and development of new symptoms as reasons. Women who continued to use ES did not report return of menopausal symptoms after the conversion. Our evidence supports the conclusion that the substitution of Estratab tablets for Premarin tablets among users of ERT was well tolerated and acceptable to most affected women in this HMO setting.

Several excretory pathways for cholephilic compounds have been known. To examine the changes in excretory pathways in cholestasis induced by ethinyloestradiol, various bile acids, organic anions and organic cations were intravenously administered to ethinyloestradiol-treated rats and their biliary excretion was studied. Biliary excretion of taurocholate was slightly delayed, but its excretory maximum was markedly decreased. Biliary excretion of lithocholate-3-O-glucuronide, leukotriene C4, sulphobromophthalein and pravastatin was markedly impaired to a similar extent. Biliary excretion of vinblastine, a P-glycoprotein substrate, was increased, suggesting increased expression of P-glycoprotein. In contrast, biliary excretion of erythromycin, a cationic antibiotic, was markedly impaired. In conclusion, ethinyloestradiol treatment altered the biliary excretion of organic compounds, which may partly be related to changes of the canalicular transporters.

Background: Prospective studies have shown that doses equivalent to conjugated equine estrogens of 0.625 mg/d or higher are needed to produce a significant increase in bone mineral density of the lumbar spine., Objectives: To determine the effects of unopposed esterified estrogens on bone mineral density, lipid levels, and endometrial tissue structure, and to relate these effects to changes in plasma estradiol levels., Methods: Four hundred six postmenopausal women were given calcium, 1000 mg/d, and randomly assigned to receive continuous esterified estrogens (0.3, 0.625, or 1.25 mg/d) or placebo for 24 months. Bone mineral density measurements and endometrial and laboratory assessments were conducted every 6 months; plasma estradiol concentrations were measured after 12, 18, and 24 months., Results: All doses of esterified estrogens produced significant increases in bone mineral density of the lumbar spine compared with baseline and with placebo at 6, 12, 18, and 24 months. Mean plasma estradiol levels increased with esterified estrogens dose, and individual subject bone mineral density changes appeared related to plasma estradiol concentrations. Clinically relevant rates of endometrial hyperplasia were noted only in the groups receiving 0.625 and 1.25 mg of esterified estrogens daily. Lipid changes were dose related and apparent in all groups., Conclusions: Esterified estrogens at doses from 0.3 to 1.25 mg/d, administered unopposed by progestin, produce a continuum of positive changes on bone and lipids. Plasma estradiol concentrations increased with esterified estrogens dose and were related to positive bone mineral densities. The 0.3-mg dose resulted in positive bone and lipid changes without inducing endometrial hyperplasia.

Objective: To survey the frequency of use of certain hair-treatment products containing hormones or placenta among different racial groups attending the pediatric clinics of military medical treatment facilities., Methods: Parents/caretakers attending pediatric clinics at four U.S. Army hospital clinics were requested to complete a questionnaire listing hormone/placenta-containing hair products., Results: Of the 521 respondents, 64% of African-Americans and 6.9% of whites used products containing hormone/placenta (p < 0.0001). Of those parents who used such products, 55.5% used them on their children. An additional 5.5% of children (from a restricted sample) went to a barber and might have been exposed to hormone/placenta-containing products. Analysis of four products showed the presence of estriol and/or estradiol., Conclusions: A majority of African-Americans but very few whites use hair products containing hormone/placenta, and more than half of those who use such hair products also used them on their children. We speculate that the use of these hair products on children may effect their sexual maturation.

Objective: To compare an oral estrogen-androgen combination with estrogens alone on bone, menopausal symptoms, and lipoprotein profiles in postmenopausal women., Methods: Surgically menopausal women received oral esterified estrogens (1.25 mg), or esterified estrogens (1.25 mg) and methyltestosterone (2.5 mg) daily, for 2 years. Bone mineral density of the lumbar spine and hip, menopausal symptoms, lipoprotein profiles, and biochemical and hematologic indices were evaluated., Results: Sixty-six patients were enrolled in the study. Both treatment regimens prevented bone loss at the spine and hip; combined estrogen-androgen therapy was associated with a significant increase in spinal bone mineral density compared with baseline (n = 24; mean score +/- standard error 3.4 +/- 1.2%, P < .01). In the estrogen group, high-density lipoprotein (HDL) cholesterol increased significantly and low-density lipoprotein cholesterol decreased significantly. Cholesterol, HDL cholesterol, and triglycerides decreased significantly in the estrogen-androgen group. Menopausal symptoms of somatic origin (hot flashes, vaginal dryness, and insomnia) were improved significantly by both treatments. Neither adverse hepatic effects nor significant safety or tolerance problems were reported in either group., Conclusion: Oral estrogen-androgen increased vertebral bone mineral density compared with pre-treatment values and relieved somatic symptoms. Safety indices, including lipoprotein levels, indicated that the combination was well tolerated over the 2 years of treatment.

Objective: To compare the efficacy and safety of esterified estrogens with and without methyltestosterone., Methods: Twenty-six women participated in a double-blind randomized trial for 6 months. Outcome measures included serum total and lipoprotein-bound cholesterol, vasomotor symptoms, vaginal cytology and endometrial histology, and chemistry values. Analysis of variance and t test statistics were used to assess differences., Results: After 6 months of therapy, the treatment groups were comparable with regard to symptom scores, vaginal cytology and endometrial histology scores, and clinical laboratory test values. Treatment with esterified estrogens plus methyltestosterone significantly decreased total cholesterol, high-density lipoprotein cholesterol (HDL), HDL2, HDL3, and apolipoprotein A1 compared to esterified estrogens alone., Conclusions: Esterified estrogens with or without methyltestosterone were effective at reducing menopausal symptoms and were well tolerated over 6 months of continuous treatment. A significant decrease in cholesterol and apolipoproteins in the estrogen plus methyltestosterone group suggests a potentially adverse impact on the beneficial effect normally imparted by estrogen therapy.

In these studies of tumor cell growth interaction we have used two tumors differing from each other in sensitivity to estradiol, transplanted to opposite sites of the same nude mice. In the first experiment we found that the growth of an estradiol-sensitive tumor may be delayed by the presence of an estradiol-resistant tumor in the same animal. Although the growth pattern was changed, proliferative activity, as reflected in the S-phase fraction measured by flow cytometry, and the steroid receptor concentrations were unchanged. Increase of circulating estradiol, however, protects the estradiol-sensitive population from this down-regulation of growth. Findings in a second experiment suggest that this growth delay is probably caused by changes such as decrease in labelling index, increase of non BrdU-incorporating cells in the S-phase, and cell loss in estradiol-sensitive tumors. We concluded that the estradiol-resistant tumor population may secrete some factor(s) acting as endocrine product(s) which may delay the growth of estradiol-sensitive cell populations when the tumors are grown in an estradiol-poor environment. If our model also represents interactions between tumor subpopulations within a single tumor, these findings may have implications for our understanding of the biology of tumor progression in some hormone-related human tumors.

The specific uptake and distribution of steroidal and non-steroidal [77Br]bromovinylestrogens were studied in immature female rats to assess the potential of these radioligands for imaging or therapy of estrogen receptor (ER) positive cancers. E-17 alpha [77Br]bromovinylestradiol, its 3 methyl ether and 11 beta-methoxy derivative, as well as the triphenylethylene estrogen, 1,1-bis[4-hydroxy-phenyl]-2-[77Br]bromo-2- phenylethylene all showed diethylstilbestrol inhibitable, specific uptake of radiobromine between 2 and 16 hr after i.p. administration. The highest concentrations in the estrogen target tissues and the highest target tissue-to-blood ratios were found with E-17 alpha-[Br]bromovinyl-11 beta-methoxyestradiol, but it also had rather high nonspecific uptake in all tissues. The triphenylethylene estrogen showed comparable specific uptake in estrogen target tissues to 17 alpha [77Br]bromovinylestradiol at 2 hr but better apparent retention, indicated by higher specific target tissue levels at the later time points. Thus, [77Br]bromovinyl-11 beta- methoxyestradiol and 1,1-bis[4-hydroxyphenyl]-2-[77Br]bromo-2-phenylethylene appear most favorable for these applications.

We describe a simple method for extraction of estrogens that is efficient over the whole polarity range of estrogen metabolites in urine and plasma. A small reversed-phase C18 cartridge (Sep-Pak C18, Waters Associates, Inc.) is used for this purpose. After application of the biological fluid, followed by an aqueous wash, estrogens are eluted in 3 mL of methanol. For urine the pH should be kept at 3 before the methanol elution. When estrogens are extracted from plasma, the steroid-protein interaction is counteracted by the presence of triethylammonium sulfate and D-norgestrel at pH 5. The recoveries of added 3H-labeled estrogens are essentially quantitative. The capacity of the column is high and estrogens can be extracted from large volumes of pregnancy urine without loss in the washing water. Because of the high flow rate (2 mL/min) and the small volume of methanol in which the estrogens are eluted, the whole procedure is convenient and rapid.

Monoiodohexestrol exhibits 10 to 15% specific binding to the 8S estrogen receptor while the remainder binds to nonreceptor 4S proteins. Reduction of nonreceptor binding with either thyroxine or 8-anilino-1-naphthalene sulfonic acid was not quantitative. Thus no accurate determination of the concentration of receptor sites in the radioreceptor assay was possible by graphical analysis. Two additional estrogens--17 alpha [125I]iodoethynylestradiol and 17 alpha-[125I]iodoethynyl-11 beta-methoxy estradiol--were synthesized at high specific activity. Although the iodoethynyl derivatives were stable under synthetic conditions, deiodination in the presence of proteins is too fast to allow either in vivo or in vitro use. To make these compounds clinically useful, therefore, chemical modification to reduce nonreceptor binding and the rate of dehalogenation must be undertaken.

Propargyl amine was protected by condensing it with 2,5-hexane-dione to give 2,5-dimethyl-N-(2'-propyn-1'-yl)pyrrole (2). The latter was converted to the corresponding Grignard reagent with ethylmagnesium bromide, and then condensed with estrone tetrahydropyranyl ether to give 17 alpha-[3'-(2'',5''-dimethyl-1''-pyrryl)-1'-propyn-1'-yl)-1,3 ,5( 10)- estratriene-3,17 beta-diol 3-tetrahydropyranyl ether (3), in 85% yield. Acetic acid and methanol cleaved the tetrahydropyranyl ether group, and hydroxylamine and sodium bicarbonate cleaved the pyrrole ring to give 17 alpha-(3'-amino-1'-propyn-1'-yl)-1,3,5(10)-estratriene-3,17 beta-diol (1), estrynamine. Several derivatives and analogs of 1 were also synthesized. Estrynamine binds to estrogen receptor with an RBA of 0.0045 (estradiol = 1.0). Several of the compounds, including estrynamine, are weak estrogens (stimulation of prolactin synthesis).

The mass spectral fragmentation pattern and the relative intensities of the fragment peaks of the D ring in some 8-dehydroheteroaromatic steroids were studied. Characteristic differences for the cis and trans isomers were ascertained and were used for the determination of the stereochemistry of thiasteroids of which only one isomer was obtained.

Some DDT analogs are estrogenic, particularly o,p'-DDT, which comprises approximately 15-20% of the commercial DDT mixture. Whether this compound or its metabolites are active has not been established. In fact, the data obtained thus far are more confusing than enlightening. For example, CCl4 pretreatment of immature female rats has been reported to inhibit or enhance estrogenic activity of o,p'-DDT, and SKF-525A pretreatment has been reported to enhance or not alter the estrogenic effect. Although o,p'-DDT inhibits binding of estradiol to the estrogen receptor from rat or human at low levels (approximately 1-10 micrometer) in vitro, higher levels are required to inhibit nuclear binding of [3H] estradiol in incubated whole uteri. Futhermore, o,p'-DDT appears to be neither estrogenic nor antiestrogenic in an in vitro estrogen assay. Methoxychlor appears to be "activated" by metabolism, and it is probable that phenolic metabolites are responsible for its estrogenic activity. Since chlorinated hydrocarbons often enhance the metabolism of steroids and may reduce circulating levels of steroids, interactions of the exogenous hormonally active agents with steroid receptors may be self-potentiating in vivo.

During the rat estrous cycle, estradiol (E2) and progesterone (P) synergize to activate reproductive behavior. However, receptivity and proceptivity can be elicited by E2 alone in ovariectomized (OVX) animals, particularly when E2 doses are high. The purpose of this study was to determine the neuroendocrine mechanism by which E2 elicits P-dependent reproductive behavior. Adult OVX females received estrogen treatment for 72 h, which consisted of 5 mm Silastic capsules containing 100% E2 or 10% E2, or of 3 injections of estradiol benzoate (EB; 20 micrograms daily). At 72 h, animals were sacrificed for nuclear progestin receptor (NPR) measurements, while others were tested for reproductive behavior. The remaining animals received 1-mg injections of E2, P, moxestrol (Mox) or oil, and either were sacrificed 2 h later for NPR measurements or were tested 4 h later for reproductive behavior. A subset of the animals receiving 1 mg E2 received concurrent administration of the protein synthesis inhibitor, anisomycin (ANI; 100 mg/kg). Acute administration of 1 mg of E2 or P significantly elevated proceptivity, receptivity and NPRs in the mediobasal hypothalamus-preoptic area (MBH-POA) and pituitary (PIT) in females primed with 100% E2. An equivalent dose of Mox was without effect. ANI blocked the acute activation of feminine reproductive behavior by 1 mg of E2. In the absence of acute steroid administration, animals primed for 72 h with EB showed higher levels of reproductive behavior than animals primed with 100% E2.(ABSTRACT TRUNCATED AT 250 WORDS)