Physiological Estrogen Research Articles

Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells

BackgroundOur study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line.MethodsFor each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens.ResultsIndividual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen’s response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose–response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the vehicle control, suggesting a possible apoptotic response. Extrinsic caspase 8 activity was suppressed by estradiol, elevated by bisphenol S, and unaffected by mixtures. Intrinsic caspase 9 activity was inhibited by estradiol, and by xenoestrogen combinations (at 10-14 and 10-8 M). Mixtures of xenoestrogens impeded the estradiol-induced release of prolactin.ConclusionsIn mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

Bisphenol S Disrupts Estradiol-Induced Nongenomic Signaling in a Rat Pituitary Cell Line: Effects on Cell Functions

Background: Bisphenol A (BPA) is a well-known endocrine disruptor that imperfectly mimics the effects of physiologic estrogens via membrane-bound estrogen receptors (mERα, mERβ, and GPER/GPR30), thereby initiating nongenomic signaling. Bisphenol S (BPS) is an alternative to BPA in plastic consumer products and thermal paper.Objective: To characterize the nongenomic activities of BPS, we examined signaling pathways it evoked in GH3/B6/F10 rat pituitary cells alone and together with the physiologic estrogen estradiol (E2). Extracellular signal-regulated kinase (ERK)– and c-Jun-N-terminal kinase (JNK)–specific phosphorylations were examined for their correlation to three functional responses: proliferation, caspase activation, and prolactin (PRL) release.Methods: We detected ERK and JNK phosphorylations by fixed-cell immunoassays, identified the predominant mER initiating the signaling with selective inhibitors, estimated cell numbers by crystal violet assays, measured caspase activity by cleavage of fluorescent caspase substrates, and measured PRL release by radioimmunoassay.Results: BPS phosphoactivated ERK within 2.5 min in a nonmonotonic dose-dependent manner (10–15 to 10–7 M). When combined with 10–9 M E2, the physiologic estrogen’s ERK response was attenuated. BPS could not activate JNK, but it greatly enhanced E2-induced JNK activity. BPS induced cell proliferation at low concentrations (femtomolar to nanomolar), similar to E2. Combinations of both estrogens reduced cell numbers below those of the vehicle control and also activated caspases. Earlier activation of caspase 8 versus caspase 9 demonstrated that BPS initiates apoptosis via the extrinsic pathway, consistent with activation via a membrane receptor. BPS also inhibited rapid (≤ 1 min) E2-induced PRL release.Conclusion: BPS, once considered a safe substitute for BPA, disrupts membrane-initiated E2-induced cell signaling, leading to altered cell proliferation, cell death, and PRL release.

What are the physiological estrogens?

Estradiol (E2) is the principal physiological estrogen in mammals. E2 and its active metabolites, estrone and estriol have a characteristic phenolic A ring, unlike progesterone, testosterone, cortisol and aldosterone, which have an A ring containing a C3-ketone, a Δ4 bond and a C19 methyl group. Crystal structures of E2 in the estrogen receptor (ER) confirm the importance of the A ring in stabilizing E2 in the ER. However, other steroids, including Δ5-androstenediol, 5α-androstanediol and 27-hydroxycholesterol, which have a saturated A ring containing a 3β-hydroxyl and a C19 methyl group, also mediate physiological responses through binding to estrogen receptor-α (ERα) and ERβ. Moreover, selective estrogen response modulators (SERMs) with diverse structures also regulate transcription of ERα and ERβ. Our understanding of the physiological responses mediated by these “alternative” estrogens is in its infancy. Further studies of the role of these steroids and SERMs in regulating responses mediated by ERα and ERβ a variety of tissues, during different stages of development, are likely to uncover additional estrogenic activities.

Regiospecificity and Stereospecificity of Human UDP-Glucuronosyltransferases in the Glucuronidation of Estriol, 16-Epiestriol, 17-Epiestriol, and 13-Epiestradiol

The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues' difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher V(max) values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones.

Phytoestrogens and soy products: perspectives of application

Isoflavones and their metabolites are termed phytoestrogens because they bind to estrogen receptors, although weakly compared to physiologic estrogens. Soybean is the main source of phytoestrogens. Several recent reviews concluded that there is no evidence that phytoestrogens relieve menopausal symptoms better than placebo. At the same time, some studies suggest their efficacy. In theory, the use of phytoestrogens for hormone replacement appears irrational: biological action of estrogens is receptor-mediated; the question is therefore, why the vegetable analogues should be used instead of physiological hormones optimally complementary to the receptors. Apparently, the problem should be seen within the scope of placebo marketing under the guise of evidence-based medications. For example, a supposed anti-atherogenic effect of phytoestrogens was confirmed by doubtful experiments with cell cultures, which are discussed here. Furthermore, there is a contradiction: phytoestrogens are supposed to compensate for estrogen deficiency in menopause; but at the same time, their estrogenic potential does not prevent the widespread use of soy for infant food and other foodstuffs. Environmentally relevant doses of phytoestrogens have impacts on ovarian differentiation, fertility and genderrelated behavior in animals. In conclusion, the beneficial and potentially harmful effects of phytoestrogens should be clarified by independent research, which can be of importance for the future of the soybean in agriculture.

Low Estrogen Exposure and/or Defective Estrogen Signaling Induces Disturbances in Glucose Uptake and Energy Expenditure

Correlations between estrogen signaling and human morbidity seems to be very unclear and difficult topic. Nowadays, the opinions of scientists have been partially changed regarding “estrogen induced diseases”, but the overwhelming literary data are still contradictory. Physiological estrogen levels in healthy premenopausal women supply protection against insulin resistance, hypertension, cardiovascular diseases and malignancies as compared with men of the same age. However, after menopause a decreased ovarian estrogen synthesis will rapidly deteriorate the glucose sensitivity and increases the prevalence of life threatening diseases. New findings on the functions of tissular estrogen receptors (ERs) indicate that estradiol plays important role in the maintenance of glucose homeostasis and energy expenditure. Diverse disorders associated with insulin resistance are usually well treatable by estradiol substitution both in pre- and postmenopausal women as well as in animal experiments. ERs seem to have balanced interplay in the maintenance of adaptation to the momentarily changing intra and extracellular stimuli. This equilibrium may be shattered in case of a defective estrogen supply or by the derangement of ER signaling pathways. Understanding the crosstalk and interplay between ERs illuminates the fact that there is no good or bad ER isoform, but they construe a complex system so as to achieve an ideal internal milieu. Considering the regulatory effects of ERs on food intake, insulin secretion, glucose uptake and metabolic processes, estrogen administration may be a therapeutic avenue to repair insulin sensitivity in patients with dysmetabolism and diabetes mellitus.

G protein-coupled estrogen receptor1 (GPER1) may mediate Rho-kinase (ROCK-2) up-regulation in coronary endothelial cells

Effect of estrogenic compounds and 17β-estradiol (E2), which induces endothelial cell motility, was investigated on ROCK-2 expression in rat coronary vascular endothelial cells (CVEC). The CVEC were isolated from the heart of Wistar rats by collagenase (0.04%) and incubated with E2 (1-100 nM), estrogen receptor α (ERα) agonist: propyl pyrazole triol (PPT, 10 nM); ERβ agonists: (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN, 10 nM) and E2-conjugate with bovine serum albumin (E2-BSA, 1 nM); and GPER1 agonist: G1 (100 nM). Furthermore, the effect of combination of E2 with estrogen receptors (ERs) antagonist and GPER1 agonist, ICI-182780 (10 µM), physiological estrogen antagonists: progesterone (P4, 10-100 nM) and testosterone (T, 10-100 nM); transcription inhibitor: actinomycin-D (1 µg/ml); GPER1 antagonist: G-15 (100 nM), superoxide dismutase, (SOD, 500 U/ml); Gi/o protein inhibitor: pertussis toxin (PTX, 100 µg/ml); and epidermal growth factor receptor (EGFR) blocker: AG-1478 (10 µM) was tested. After 24h incubation, ROCK-2 and GPER1 protein expressions were detected in the CVEC by Western-blotting. E2, ICI-182780, and G1 but not E2-BSA significantly up-regulated ROCK-2 expression, which was suppressed by actinomycin-D, PTX, AG-1478, and G-15. However, PPT and DPN had no effects on the ROCK-2 expression. ICI-182780, P4, T or SOD did not antagonize the E2 action. GPER1 expression was demonstrated in the CVEC. Estrogens could up-regulate ROCK-2 in the rat CVEC through GPER1 and EGFR transactivation.

A main role for metabotropic glutamate receptor 1 in the neuroprotective effect of estrogen

Estrogen exerts neuroprotective activity under different experimental conditions through classical nuclear receptors, but mainly receptors expressed at the cell surface. Transducing mechanisms activated by these membrane estrogen receptors in the brain have been intensely investigated and, among others, interaction with G-protein coupled, metabotropic glutamate (mGlu) receptors has been considered. Besides mediating physiological estrogen functions, such as regulation of hormone production or sexual behavior in the hypothalamus, mGlu receptors, specifically mGlu1 receptor subtype, take part to the protective effect of estrogen in a model of neuronal toxicity induced by β-amyloid peptide. Coupling of estrogen receptor to mGlu1 receptor is supported by co-immunoprecipitation, similar neuroprotective effect induced by either receptor activation, lack of additivity when the two receptors are activated at the same time and prevention of the protective effect when antagonists of the other receptor are used, i.e. reduction of the protective effect of estrogen by the mGlu1 receptor antagonist and vice versa. In addition, the phosphatidylinositol-3 kinase/Akt pathway may represent the common signaling pathway to produce neuroprotection. These data introduce a novel view of the mechanisms underlying the neuroprotective activity of estrogen and open new perspectives also for future pharmacological interventions. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:150%; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

A Mechanistic Study of the Effect of Doxorubicin/Adriamycin on the Estrogen Response in a Breast Cancer Model

Objective: Estrogen treatment limits the cytotoxic effects of chemotherapy in estrogen receptor-positive (ER+) breast cancer cell lines, suggesting that estrogen pathway signaling may confer chemotherapeutic resistance. This study investigates the molecular responses of ER+ breast cancer cell lines to the chemotherapeutic agent, doxorubicin, in the presence or absence of estrogen. Methods: ER+ MCF-7 and T47-D cells were cultured in hormone-starved or estrogen-containing media with or without doxorubicin at concentrations mimicking the low concentrations seen in plasma and tumor microenvironments in humans following typical bolus administration. Protein levels, phosphorylations, and interactions of estrogen-signaling molecules were assessed following these treatments, as well the effects of ER signaling inhibitors on cell proliferation. Results: Surprisingly, estrogen and doxorubicin co-treatment markedly induced pro-growth alterations compared to doxorubicin alone and modestly enhanced estrogen alone-induced changes. Several inhibitors suppressed cell proliferation in the presence of doxorubicin and estrogen. Conclusions: These findings demonstrate that molecular changes caused by doxorubicin in ER+ breast cancer cells can be reversed by estrogen, providing molecular evidence for the poorer responses of ER+ tumors to doxorubicin in the presence of physiologic estrogen levels. Our results also suggest that the addition of drugs targeting the ER, EGFR, the SFKs, MEK, PI3K, and/or the MMP proteins to a conventional chemotherapy regimen may improve chemosensitivity.

Non-Genomic Effects of Xenoestrogen Mixtures

Xenoestrogens (XEs) are chemicals derived from a variety of natural and anthropogenic sources that can interfere with endogenous estrogens by either mimicking or blocking their responses via non-genomic and/or genomic signaling mechanisms. Disruption of estrogens’ actions through the less-studied non-genomic pathway can alter such functional end points as cell proliferation, peptide hormone release, catecholamine transport, and apoptosis, among others. Studies of potentially adverse effects due to mixtures and to low doses of endocrine-disrupting chemicals have recently become more feasible, though few so far have included actions via the non-genomic pathway. Physiologic estrogens and XEs evoke non-monotonic dose responses, with different compounds having different patterns of actions dependent on concentration and time, making mixture assessments all the more challenging. In order to understand the spectrum of toxicities and their mechanisms, future work should focus on carefully studying individual and mixture components across a range of concentrations and cellular pathways in a variety of tissue types.

Synthesis and Biological Evaluation of the Salicylamide and Salicylic Acid Derivatives as Anti-Estrogen Agents

Alkylphenols have xenoestrogenic activity, which mimic the action of physiological estrogens and these mimicking activities are mainly mediated by nongenomic pathway. Nongenomic pathway plays a pivotal role in breast, endometrial and ovarian cancers' growth and development. In this study, various alkylphenol derivatives were prepared and screened for their anti-uterotrophic and uterotrophic activity. Among these compounds, 2-hydroxy-5-nonanoylbenzamide (compound 1b) showed 93.99% inhibitory activity in the anti-uterotrophic test performed, and was found inactive in the uterotrophic activity test. Moreover, all test compounds were examined for the effect on uterine histopathological changes, and plasma 17β-estradiol (E2) level. Compound 1b was also tested for in vitro anti-cancer activity against ER+, human breast cancer cell line MCF-7, and it reduced cell viability to 74.01% at 50 nM concentration.

Neuropeptide Y antagonist improves bone density in ovariectomized female rats

Neuropeptide Y plays a key role in the physiological control of energy homeostasis. The role of neuropeptide Y in ovariectomized animals has not been fully established. Multiple neuropeptide Y receptor subtypes are thought to mediate neuropeptide Y activity in both energy regulation and bone metabolism. We examined the role of neuropeptide Y(1) on bone density and strength in adult ovariectomized female rats compared with estrogen administration. A sustained‐dose of a neuropeptide Y(1) receptor antagonist (0.1 μg/day) reduced body weight similar to animals receiving sustained physiologic estrogen levels. In addition to body weight reductions, bone densities were increased and bone strength was improved over ovariectomized animals that were untreated. The histological and biomechanical evaluations showed Y(1) receptor antagonist had similar improvements compared with estrogen treated animals. These findings suggest neuropeptide Y(1) may play a role at the central nervous system and ultimately regulate energy homeostasis and bone metabolism.

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture.

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Influence of gender and estrous cycle on plasma and renal catecholamine levels in rats

Several studies have demonstrated that gonadal hormones show significant effects on the brain and signaling pathways of effector organs/cells that respond to neurotransmitters. Since little information is available concerning the impact of male and female gonadal hormones on the renal and peripheral sympathetic system, the objective of this study was to further assess whether and how the renal content and plasma concentration of catecholamines are influenced by gender and the estrous cycle in rats. To achieve this, males Wistar rats were divided into 4 groups: (i) sham (i.e., control), (ii) gonadectomized, (iii) gonadectomized and nandrolone decanoate replacement at physiological levels or (iv) gonadectomized and nandrolone decanoate replacement at high levels. Female Wistar rats were divided into 6 groups: (i) ovariectomized (OVX), (ii) estrogen replacement at physiological levels and (iii) estrogen replacement at at high levels, (iv) progesterone replacement at physiological levels and (v) progesterone replacement at at high levels, and (vi) sham. The sham group was subdivided into four subgroups: (i) proestrus, (ii) estrus, (iii) metaestrus, and (iv) diestrus. Ten days after surgery, the animals were sacrificed and their plasma and renal catecholamine levels measured for intergroup comparisons. Gonadectomy led to an increase in the plasma catecholamine concentration in females, as well as in the renal catecholamine content of both male and female rats. Gonadectomized males also showed a lower level of plasma catecholamine than the controls. The urinary flow, and the fractional excretion of sodium and chloride were significantly increased in gonadectomized males and in the OVX group when compared with their respective sham groups.

Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I

Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the Gαi subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of Gα protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated Gαi at 15–30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of Gαi for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, Gαs was neither activated nor deactivated by these treatments with estrogens. ERα and Gαi co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with Gαi and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. Conclusions: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with Gαi and caveolin I, but with some different characteristics, which could explain their disruptive actions.

Protective effects of physiological doses of estrogen during hepatocarcinogenesis induced by diethylnitrosamine and N-nitrosomorpholine.

Concern that much controversy exists with respect to the role of estrogen in hepatocarcinogenesis prompted us to examine the effect of estrogen, at physiological concentrations, on our established HCC rat model induced by diethylnitrosamine and N-nitrosomorpholine. Female Sprague-Dawley rats were randomly divided into four groups (Group 1: Control, Group 2: Sham-operated, Group 3: Ovariectomy, Group 4: ovariectomy+estrogen) with treatment of a single i.p. injection of diethylnitrosamine (100mg/ kg body weight) followed by N-nitrosomorpholine (100ppm) in drinking water for 20 weeks for the established rat HCC model. Physiological estrogen was administered by 17α-Ethynylestradiol at a dose of 30μg/ kg body weight while rats in the sham-operated group were treated with saline after initiation of liver carcinogenesis. Treatment of ovariectomized animals with 17α-Ethynylestradiol (30μg/kg body weight/ day) resulted in a significant decrease in the initiation, development and metastasis of HCC and an increase in the survival time of animals dead before the termination of experiment as compared with rats treated with ovariectomy only (p<0.05); whereas this difference disappeared when compared with the other three groups. These findings show for the first time that estrogen, at physiological concentrations may reveal a protective role in hepatocarcinogenesis.

Effects of endogenous and exogenous estrogens on vaginal mucosal histology

The vaginal epithelium undergoes remarkable changes during menopause, presumably due to the physiologic estrogen reduction. Surprisingly few studies however, have quantified the histologic changes associated with estrogen supplementation. The current study was undertaken to investigate basic histologic differences between pre- and postmenopausal women and compare those with postmenopausal women using exogenous estrogen thus providing insight into both endocrinologic and age-related changes in vaginal epithelium. Analysis of histologic specimens discarded following vaginal surgery. Specimens of vaginal epithelium were obtained after prolapse surgery from premenopausal women (PRE, n = 8), postmenopausal women (POST, n = 8) and postmenopausal women using vaginal estrogen cream (EST, n = 8). After formalin fixation tissues were embedded in paraffin and sectioned and stained with Hematoxylin/Eosin or Periodic Acid Schiff. The slides were analyzed for epithelial thickness, number of epithelial layers and glycogen content. Measurements were done by 2 blinded assessors. Intraclass correlation coefficients (SPSSS 19) were used to assess interobserver agreement. Means were compared using ANOVA. The intraclass correlation coefficient between the two blinded observers for measurements of epithelial thickness in the was .921 (P < .001). The correlation between observers of the number of epithelial layers counted was not significant. Mean epithelial thickness was 167μm, 332μm and 298μm in POST, PRE and EST, respectively (P <0.05). The reduction in glycogen in the POST samples was not seen in the EST samples. Measurement of epithelial thickness is a more objective method of assessment of vaginal epithelial thickness than counting epithelial layers. Postmenopausal samples have the thinnest epithelia and the lowest glycogen content. In postmenopausal women the use of vaginal estrogen cream is associated with restoration of vaginal epithelial thickness and glycogen content.

Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases

Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17β-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17β-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differs considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the K m value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B.

Influence of estrogen on pulmonary arterial hypertension: role of oxidative stress

Pulmonary arterial hypertension (PAH) is a disease that increases the pulmonary vascular resistance, causing hypertrophy and subsequent right heart failure. Oxidative stress is involved in the pathogenesis of PAH, and estrogen is considered an antioxidant. Thus, the aim of this study was to test the hypothesis that estrogen could attenuate PAH by modulating oxidative stress. Female Wistar rats were ovariectomized or suffered the surgery simulation (sham). After 7 days, subcutaneous pellets with 17β-estradiol or sunflower oil were implanted. At this time, PAH was induced by means of a single dose of monocrotaline (MCT) (60 mg·kg(-1) i.p.). The experimental groups were as follows: (1) sham, (2) sham + MCT, (3) ovariectomy (O), (4) ovariectomy + MCT (OM), (5) ovariectomy + estrogen replacement + MCT (ORM). Hemodynamic measurements were performed 21 days after MCT or saline. Nonovariectomized animals were assessed in the stage of diestrus. Afterwards, the rats were killed to collect the heart, the lung and the liver to evaluate morphometry. Samples of the right ventricle were used to analyse the reduced glutathione : oxidized glutathione ratio. Lung congestion in the OM group, which was decreased in the ORM group, was observed. Right ventricle end-diastolic pressure was increased in the OM and the ORM groups. The glutathione ratio decreased in the groups O, OM and ORM. The data suggest that estrogen can exert great influence on the cellular redox balance. The maintenance of physiological estrogen levels may help to avoid the appearance of pulmonary oedema, characteristic of this model of PAH, and right ventricular failure.

Growth Hormone Plus Childhood Low-Dose Estrogen in Turner Syndrome

Characteristic features of Turner syndrome include short stature and ovarian failure. There is considerable evidence from nonrandomized studies that treatment with recombinant human growth hormone increases adult stature in patients with Turner syndrome. It is commonly used in such patients despite the lack of objective data from randomized, double-blind, placebo-controlled studies. Ovarian failure associated with Turner syndrome is treated with estrogen-replacement therapy. It has been proposed that very low-dose physiologic estrogen replacement during childhood combined with growth hormone therapy could provide an additional benefit on adult height than that achieved with growth hormone therapy alone. The aim of this double-blind, placebo-controlled trial was to investigate the independent effect of growth hormone therapy alone and a regimen of growth hormone combined with early ultra–low-dose estrogen on adult height in girls with Turner syndrome. A total of 149 girls (5.0–12.5 years of age) were randomized to receive 1 of 4 regimens: (1) placebo injection plus childhood oral placebo (n = 39), (2) placebo injection plus childhood oral low-dose estrogen (n = 40), (3) growth hormone injection plus childhood oral placebo (n = 35), and (4) growth hormone injection plus childhood oral low-dose estrogen (n = 35). Doses of growth hormone (0.1 mg per kg of body weight) were injected 3 times per week. Ethinyl estradiol or placebo was administered in oral doses, with adjustment for chronologic age and pubertal status. From the first visit after the 12th birthday, escalating doses of ethinyl estradiol were administered to patients in all groups, according to an escalating dosage regimen. Patients were assessed at 6-month intervals. Growth hormone therapy was continued until patients reached their protocol-specified adult height (defined as the last height measured once the height velocity was less than 1.5 cm per year). For adult height attained at 17.0 ± 1.0 years after a mean study period of 7.2 ± 2.5 years, the mean standard deviation scores for the 4 groups were as follows: −2.81 ± 0.85 for the double-placebo group, −3.39 ± 0.74 for the estrogen-alone group, −2.29 ± 1.10 for the growth hormone-alone group, and −2.10 ± 1.02 for the combined growth hormone-estrogen group (P < 0.001 among the 4 groups). Patients treated with growth hormone attained greater adult height compared with placebo (mean difference in standard deviation score, 0.78 ± 0.13, equivalent to about 5.0 cm; P < 0.001). There was a trend toward a modest synergistic growth benefit in the combined growth hormone-estrogen group compared with the estrogen-alone group; the adult height gain with combined therapy was 0.32 ± 0.17 standard deviation score; P = 0.059. These findings provide objective evidence from a placebo-controlled trial that growth hormone treatment initiated in mid-childhood increases adult height in girls with Turner syndrome. The data also suggest that the combination of ultra–low-dose childhood estrogen replacement and growth hormone therapy may improve the height gain achieved with growth hormone alone.