Estrogen In Tissues Research Articles

Novel insight in estrogen homeostasis and bioactivity in the ACI rat model of estrogen-induced mammary gland carcinogenesis.

Despite being widely used to investigate 17β-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28-42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.

A diet containing high- versus low-daidzein does not affect bone density and osteogenic gene expression in the obese Zucker rat model.

Phytoestrogens are nonsteroidal plant compounds with similar chemical structures to mammalian estrogen capable of mimicking the effect of estrogen in selective tissues. A diet rich in phytoestrogens is associated with a variety of health benefits including decreased risks for heart disease, breast cancer, and osteoporosis. Obesity has long thought to be associated with improved bone density due to increased mechanical loading, but recent literature suggests obesity may actually decrease bone health. Daidzein, a soy-derived phytoestrogen, has been shown to improve parameters of bone health in lean animal models of osteoporosis but has not been tested in obese animals. Following a one-week acclimation to a standard AIN-93G diet, 19 five-week-old female obese Zucker rats (OZR) were randomly assigned to a modified AIN-93G diet containing either high daidzein (HD, 0.121 g kg-1 feed) or low daidzein (LD, 0.01 g kg-1 feed). After 8 weeks, tibias and femurs were removed to assess true density (Archimedes principal), mechanical strength (three-point bending test), and femoral osteogenic gene expression. Serum was collected to assess osteocalcin and deoxypyridinoline. Our results indicated that there were no significant differences between the measures for tibial or femoral true density or mechanical strength for the rats in the HD and LD diet groups. Similarly, there were no significant differences in gene expressions related to osteogenic pathways, or serum biomarkers of bone formation and resorption. Overall, an increased dose of daidzein from soy protein supplementation does not elicit an improvement in markers of bone health in obese Zucker rats.

Ethnic differences in regional adipose tissue oestrogen receptor gene expression.

Studies have shown ethnic differences in body fat distribution, characterised by greater peripheral and less central fat accumulation in black compared to white South African (SA) women. As sex hormones play an important role in body fat distribution, our study aimed to determine whether differences in body fat distribution between black and white SA women were associated with subcutaneous adipose tissue (SAT) expression of oestrogen receptors (ERA and ERB) and aromatase (CYP19A1). Body fat distribution (DXA and CT) and ERA, ERB and CYP19A1 expression in abdominal and gluteal SAT were measured in 26 black and 22 white SA women. Abdominal SAT ERA and ERB did not differ by ethnicity or BMI. Gluteal ERA was higher (1.08 ± 0.06 vs 0.99 ± 0.05, P < 0.001) and ERB was lower (0.99 ± 0.06 vs 1.10 ± 0.07, P < 0.001) in black vs white SA women. CYP19A1 increased with obesity in all depots (P < 0.001). In both black and white SA women, gluteal ERA was associated with lower central fat mass (FM) and greater gynoid FM (P < 0.05), while the inverse association was shown for CYP19A1 in all depots (P < 0.01). In conclusion, ethnic differences in gluteal ERA expression were associated with differences in body fat distribution previously reported between black and white SA women.

Advances in the research of gastric signet ring cell carcinoma

Epidemiological studies in recent years have found that the incidence of gastric signet ring cell carcinoma (SRCC) has increased significantly. In this paper, we first reviewed cell origin and biomarkers of SRCC, and the relationship between SRCC and various pathological types of gastric cancer. The early diagnosis rate of gastric SRCC is low, which may be due to the unclear mechanism of pathology and precancerous lesions. In clinical practice, SRCC has the characteristics of low differentiation and high degree of malignancy. Most of patients with gastric cancer Borrmann IV(diffuse infiltrative type) are gastric SRCC, and their prognosis is poor. The average age of gastric SRCC was 55 to 61 years old. Besides, in female, the incidence of SRCC was significantly higher than that of non-SRCC gastric cancer. It is found that the expressions of estrogen and progesterone receptors in SRCC tissues are high. The relationship between gastric SRCC and sex hormones may be the cause of gender differences in the pathogenesis of gastric SRCC. Due to the low risk of lymph node metastasis in early SRCC, endoscopic mucosal resection and endoscopic submucosal dissection can be performed for <3 cm, submucosal invasive, medium differentiated tumors, or <3 cm, highly differentiated, ulcerative and submucosal lesions. For non-metastatic advanced gastric SRCC, surgical resection and adequate lymph node dissection should be performed owing to the high risk of lymph node metastasis. Adjuvant chemotherapy is also considered to improve the long-term prognosis of patients. Taxane therapy may be more effective in gastric SRCC. Recent data show that gastric SRCC and diffuse gastric cancer are more sensitive to mitomycin C, doxorubicin and docetaxel than intestinal type gastric cancer, but are not sensitive to fluorouracil and cisplatin. These treatment perspectives still need to be confirmed in future studies.

Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery.

Our understanding of the intracrine (or local) regulation of estrogen and other steroid synthesis and degradation expanded in the last decades, also thanks to recent technological advances in chromatography mass-spectrometry. Estrogen responsive tissues and organs are not passive receivers of the pool of steroids present in the blood but they can actively modify the intra-tissue steroid concentrations. This allows fine-tuning the exposure of responsive tissues and organs to estrogens and other steroids in order to best respond to the physiological needs of each specific organ. Deviations in such intracrine control can lead to unbalanced steroid hormone exposure and disturbances. Through a systematic bibliographic search on the expression of the intracrine enzymes in various tissues, this review gives an up-to-date view of the intracrine estrogen metabolisms, and to a lesser extent that of progestogens and androgens, in the lower female genital tract, including the physiological control of endometrial functions, receptivity, menopausal status and related pathological conditions. An overview of the intracrine regulation in extra gynecological tissues such as the lungs, gastrointestinal tract, brain, colon and bone is given. Current therapeutic approaches aimed at interfering with these metabolisms and future perspectives are discussed.

Activation of estrogen receptor alpha induces beiging of adipocytes

ObjectivesBrown adipose tissue (BAT) and BAT-like adipose tissues, referred to as ‘beige’ adipose tissues uncouple respiration from ATP synthesis via uncoupling protein one (UCP-1). There is a sexual dimorphism with respect to beige and BAT tissues; pre-menopausal women have more BAT and are more sensitive to BAT activation than men or postmenopausal women. We hypothesized selective activation of adipose tissue estrogen receptor alpha (ERα) induces beiging of WAT through induction of lipolysis mediated by adipose tissue triglyceride lipase (ATGL). Methods3T3-L1 and primary adipocytes were treated with the selective ERα agonist pyrazole triol (PPT), and selection deletion of ERα (using siRNA) was used to determine if selective ERα activation, or inhibition, influences the adipose tissue expression of genes associated with beiging. In a second series of experiments, ERα was selectively added back to adipose tissue of mice lacking total body ERα (ERKO) to determine if add back of ERα changed the morphology of adipose tissue to resemble beige tissues. Additionally, WT and ERKO mice were exposed to cold and FDG labeled glucose uptake was measured to determine the ability of cold to induce UCP-1 in ERKO mice. To begin to mechanistically probe how activation of ERα facilitates beiging, we tested the influence of PPT to activate the lipolytic pathway through ATGL. Finally, since ERα exerts its effects both at the genomic and non-genomic level depending on its cellular location, we determined in vivo if beiging occurs in mice expressing ERα only at the plasma membrane (MOER mice) or only at nucleus (NOER mice). ResultsSelective ERα activation by PPT increased markers of beiging in vitro in 3T3-L1 and primary adipocytes, whereas, knockdown of ERα with siRNA reduced the ability of PPT to induce beiging in vitro. ERα add back to the adipose tissue of ERKO mice resulted in multilocular adipose tissue resembling a beige phenotype. Following cold exposure, FDG labeled glucose in BAT tissues of ERKO mice was reduced when compared to weight-matched controls. Glycerol release and ATGL expression were increased after PPT treatment, while pre-treatment with the ATGL inhibitor prevented PPT's ability to increase UCP-1 expression. Finally, MOER mice were more sensitive to beiging of adipose tissues when compared to NOER mice. ConclusionOur results demonstrate for the first time that selective-activation of ERα in adipocytes increases markers of beiging and this is likely through induction of AMPK and ATGL-mediated lipolysis providing FFAs as a fuel to activate UCP-1.

In Memorium: Clara M. Szego (1916–2017)

Dr. Clara M. Szego passed away recently at the age of 101. She was a pioneer in the study of steroid hormone action, particularly the role of estrogens in target tissues. She was first to report on steroid hormone binding with proteins in the circulation in 1946. This observation led to a Ciba Foundation Award of the Endocrine Society in 1953. Claire then followed the steroid hormone and reported on interactions with membrane-associated and nuclear proteins in target cells of the reproductive tract and breast. She was also an advocate to stop the use of diethylstilbestrol (DES) in animal feed in the 1950s–1960s and later helped to develop clinical tests to identify women at risk for developing malignancy after exposure to DES in utero. Being a woman in endocrinology was challenging in the post-war period, and Claire worked to hinder gender bias in academia throughout her career. Her impact on the fields of endocrinology and steroid hormone action was profound, and the full ramifications and clinical benefits of her work remain to be realized.

Serum DHEA and Its Sulfate Are Associated With Incident Fall Risk in Older Men: The MrOS Sweden Study.

The adrenal-derived hormones dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are the most abundant circulating hormones and their levels decline substantially with age. Many of the actions of DHEAS are considered to be mediated through metabolism into androgens and estrogens in peripheral target tissues. The predictive value of serum DHEA and DHEAS for the likelihood of falling is unknown. The aim of this study was, therefore, to assess the associations between baseline DHEA and DHEAS levels and incident fall risk in a large cohort of older men. Serum DHEA and DHEAS levels were analyzed with mass spectrometry in the population-based Osteoporotic Fractures in Men study in Sweden (n = 2516, age 69 to 81 years). Falls were ascertained every 4 months by mailed questionnaires. Associations between steroid hormones and falls were estimated by generalized estimating equations. During a mean follow-up of 2.7 years, 968 (38.5%) participants experienced a fall. High serum levels of both DHEA (odds ratio [OR] per SD increase 0.85; 95% CI, 0.78 to 0.92) and DHEAS (OR 0.88, 95% CI, 0.81 to 0.95) were associated with a lower incident fall risk in models adjusted for age, BMI, and prevalent falls. Further adjustment for serum sex steroids or age-related comorbidities only marginally attenuated the associations between DHEA or DHEAS and the likelihood of falling. Moreover, the point estimates for DHEA and DHEAS were only slightly reduced after adjustment for lean mass and/or grip strength. Also, the addition of the narrow walk test did not substantially alter the associations between serum DHEA or DHEAS and fall risk. Finally, the association with incident fall risk remained significant for DHEA but not for DHEAS after simultaneous adjustment for lean mass, grip strength, and the narrow walk test. This suggests that the associations between DHEA and DHEAS and falls are only partially mediated via muscle mass, muscle strength, and/or balance. In conclusion, older men with high DHEA or DHEAS levels have a lesser likelihood of a fall. © 2018 American Society for Bone and Mineral Research.

Expression of androgen, estrogen, and progesterone hormone receptors in the penile tissues of children with different types of hypospadias.

OBJECTIVE:Androgen (AR), Estrogen (ER) and Progesterone (PR) hormones play an important role in the prenatal and postnatal development of urogenital tract and especially the penis. The expressions of AR, ER and PR receptors in penile tissues in children with hypospadiases had also been shown previously. In this leading study, to demonstrate of the sex hormone receptor expression in cases with different types of hypospadias were aimed.METHODS:This study was designed in children operated due to hypospadiases without DSD. Biopsy samples of 3 mm’s were obtained from three different sytes as the lateral parameatal tissue and the anterior corner of the prepuce, and inner layer of posterior prepuce. The presence of AR, ER and PR receptors was investigated immunehistochemically.RESULTS:Mean age was 5.4 years in 18 children with hypospadiases; in totally 33 specimens were taken in 5 subcoronal as 5 specimens, and 7 penile as 15 specimens, and 6 penoscrotal as 13 specimens. According to sytes of samples; 13 samples were from lateral para-meatal tissues, and 13 were from anterior corners of prepuces, and 7 were from inner layers of posterior prepuces. In regard to receptor expression; ER and AR receptors were positive in 29 (87.8%) and 12 (36.4%) respectively; PR receptors were negative.CONCLUSION:This study emphasized the dominant expression of estrogen receptors in penile tissues of children with hypospadias. Although there was not a manifest correlation of androgen receptors absence in regard to the severity of hypospadias patients, there was a marked estrogen receptors presence in penile tissues. These findings suggest that the disrupted androgen and estrogen receptor interaction and/or balance could play a role during the development of external genitalia in hypospadias patients. Progesterone receptor was not present and therefore the active role in the postnatal development of hypospadias is still debatable.

The Role of Dehydroepiandrosterone (DHEA) in Skeletal Muscle.

Dehydroepiandrosterone (DHEA) is a precursor of sex steroid hormones and is converted to testosterone and estradiol. Normally, androgens and estrogens produced adrenal cortex, testis, and ovary; however, recent studies revealed androgens and estrogens are synthesized by peripheral tissues such as brain, skin, liver, kidney, bone, etc. We found skeletal muscles are also capable of synthesizing androgens and estrogens from DHEA. Circulating DHEA provides substrates required for conversion into potent androgens and estrogens in peripheral tissues. Sex steroid hormone administration has important roles: one is that the enhancement of protein synthesis and anabolism, resulting in muscle growth and increased muscle strength. The other is improvement of hyperglycemia through the activation of glucose signaling pathway in skeletal muscle as well as acceleration of muscle lipid metabolism that increase peroxisome proliferator-activated receptor alpha (PPARα) and PPAR delta (PPARδ). We introduce the effect of DHEA and sex steroid hormones administration on muscle glucose and lipid metabolisms as well as the effect of sex steroid hormone on the muscle hypertrophy.

Obesity, a serious etiologic factor for male subfertility in modern society.

Obesity, defined as excessive accumulation of fat in adipose tissue, is a metabolic disorder resulting from behavioral, environmental and heritable causes. Obesity increases the risks of hypertension, diabetes, cardiovascular disease, sleep apnea, respiratory problems, osteoarthritis and cancer. Meanwhile, the negative impact of obesity on male reproduction is gradually recognized. According to the clinical investigations and animal experiments, obesity is correlated with reductions in sperm concentration and motility, increase in sperm DNA damage and changes in reproductive hormones. Several mechanisms can elucidate the effects of obesity on sperm functions and male subfertility, i.e., the excessive conversion of androgens into estrogens in redundant adipose tissue causes sexual hormone imbalance, subsequently resulting in hypogonadism. Secondly, adipokines produced by adipose tissue induce severe inflammation and oxidative stress in male reproductive tract, directly impairing testicular and epididymal tissues. Moreover, increased scrotal adiposity leads to increase gonadal heat, continuously hurting spermatogenesis. Therefore, obesity alters the systematic and regional environment crucial for spermatogenesis in testis and sperm maturation in epididymis, and finally results in poor sperm quality including decreased sperm motility, abnormal sperm morphology and acrosome reaction, changed membrane lipids and increased DNA damage. Furthermore, recent studies indicate that epigenetic changes may be a consequence of increased adiposity. A major effort to identify epigenetic determinants of obesity revealed that sperm DNA methylation and non-coding RNA modification are associated with BMI changes and proposed to inherit metabolic comorbidities across generations. This review will explain how obesity-related changes in males to influence sperm function and male fertility as well.

Characterization of serum estradiol level and tissue estrogen receptor immunostaining with clinical response and reproductive factor changes in Chinese female patients with esophageal squamous cell carcinoma

Pre-menopausal female patients have a prolonged survival than post-menopausal patients, indicating that estrogen and/or estrogen receptor (ER) may have some biological effects on prognosis. ER expression in cancer tissue has been reported to be a significant prognostic marker in multiple human cancers. However, the prognostic value of estrogen and/or ER on female patients with esophageal squamous cell carcinoma (ESCC) is rarely reported. The present study was undertaken to elucidate the associations of serum estradiol level, tissue estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) expression with clinical response and reproductive factor changes in 387 female ESCC patients. Radioimmunoassay revealed that serum estradiol level was higher in pre-menopausal than those in peri-menopausal and post-menopausal patients. Furthermore, patients with higher serum estradiol level appeared to have a better survival. Immunostaining results suggested that ERα positive (+) expression was mainly located in cytoplasm of tumor cells with a positive rate of 69.9% and ERβ (+) was mainly located in nucleus of tumor cells with a positive rate of 64.9%. We did not find the relations of ER expression with tumor invasion (P>0.05), lymph node metastasis (P>0.05), TNM staging (P>0.05) and treatment method (P>0.05). Surprisingly, ERα (+) expression was higher in post-menopausal patients than those in pre-menopausal patients (P<0.05). Patients with number of pregnancy≥4 have a higher ERβ (+) expression than those patients with≤3 (P<0.05). Univariate and multivariate survival analysis showed that ERβ (+) expression in addition to ERα (−) expression are favorable prognostic markers in female ESCC patients (P<0.05). Further related study is needed to in-depth explore the potential mechanisms of ERα and ERβ in survival of female patients with ESCC.

Simultaneous determination of UV-filters and estrogens in aquatic invertebrates by modified quick, easy, cheap, effective, rugged, and safe extraction and liquid chromatography tandem mass spectrometry

Ultraviolet-filters (UV-filters) and estrogens have attracted increased attention as contaminants of emerging concern (CECs) due to their widespread occurrence in the environment. Most of these CECs are hydrophobic and have the potential to accumulate in aquatic organisms. To date, co-analysis of UV-filters and estrogens has not been reported due, in part, to the complex environmental matrices. Here, a multi-residue method has been developed for simultaneous determination of five UV-filters and three estrogens in tissue from aquatic and marine organisms. The procedure involved a modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction with a novel reverse-solid-phase extraction (reverse-SPE) cleanup in place of dispersive-SPE, followed by liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis. The tissue mass, acetonitrile content, and salt conditions for QuEChERS extraction, along with the reverse-SPE cartridge material and elution conditions, were thoroughly investigated and optimized. Five UV-filters (i.e., 3-(4-methylbenzylidene) camphor, benzophenone-3, ethylhexylmethoxycinnamate, homosalate, and octocrylene) and three estrogens (i.e., estrone, 17β-estradiol, and 17α-ethinylestradiol) were simultaneously analyzed by taking advantage of wrong-way-round ionization in LC–MS/MS. The optimized analytical protocol exhibited good recoveries (>80%) for target compounds and enabled their detection at concentrations as low as 0.2ng/g in 50mg tissue samples. The method was applied to determine concentrations of target analytes in four invertebrates (i.e., Orconectes virilis, Procambarus clarkii, Crassostrea virginica, and Ischadium recurvum). All eight target analytes were detected at least once in the tissue samples, with the highest concentration being 399ng/g of homosalate in O. virilis. These results highlight the ubiquitous bioaccumulation of CECs in aquatic and marine invertebrates.

Estradiol-mediated improvements in adipose tissue insulin sensitivity are related to the balance of adipose tissue estrogen receptor α and β in postmenopausal women.

We recently demonstrated that short-term estradiol (E2) treatment improved insulin-mediated suppression of lipolysis in postmenopausal women, but to a greater extent in those who were late compared to early postmenopausal. In this follow-up study we tested whether subcutaneous adipose tissue (SAT) expression of estrogen receptors (ER) α and β differs between early and late postmenopausal women. We further tested whether the balance of ERα to ERβ in SAT determined the effect of E2 on SAT insulin sensitivity. The present study included 35 women who were ≤6 years past menopause (EPM; n = 16) or ≥10 years past menopause (LPM; n = 19). Fasted SAT samples were taken following 1-week transdermal E2 treatment or placebo (PL) in a random cross-over design. Samples were analyzed for nuclear/cytosolic protein content and mRNA expression using Western blot and qPCR, respectively. While ESR1 increased slightly (~1.4-fold) following E2 treatment in both groups, ERα and ERβ protein expression did not differ between groups at baseline or in response to E2. However, the balance of ERα/ERβ protein in the SAT nuclear fraction increased 10% in EPM compared to a 25% decrease in LPM women (group x treatment interaction, p<0.05). A greater proportion of ERα/ERβ protein in the nuclear fraction of SAT at baseline (placebo day) was associated with greater reduction in SAT insulin resistance (i.e., better suppression of lipolysis, EC50) in response to E2 (r = -0.431, p<0.05). In conclusion, there do not appear to be differences in the proportion of adipose tissue ERα/ERβ protein in late, compared to early, postmenopausal women. However, the balance of ERα/ERβ may be important for E2-mediated improvement in adipose tissue insulin sensitivity.Trial Registration: Clinical Trials#: NCT01605071

Inhibition of tight junction protein ZO-1 expression by estrogen in human gut mucosa

Abstract Background Females have stronger cellular and humoral immunities compared to males. Sex hormones are playing roles in these differences; however, the role of sex hormones in human gut permeability is essentially unknown. Methods The expression of tight junction ZO-1, estrogen receptor (ER)-α and β in male and female primary gut tissues were analyzed using immunohistochemistry. The inhibition of estrogen on ZO-1 was evaluated in primary gut tissues by qPCR. ZO-1 expression and NF-κB activation in Caco-2 cells was evaluated by western blot, immunofluorescent staining and reporter activity assays. Data are presented as median (interquartile range [IQR]). Results The expression of ZO-1 and ER-β but not ER-α were present in both male and female gut tissues, the median immunofluorescence intensities (MFI) of ZO-1 expression were 2.48 (2.40–3.11) and 3.59 (3.11–4.23) for females and males respectively (P = 0.02, non-parametric Mann-whitney test). No sex difference in MFI expression of ER-β was found (P = 0.79). After 24 h incubating with physiologic dose of estrogen (200 pg/mL) in primary gut tissues in vitro, the median ZO-1 mRNA relative expression to GADPH by qPCR was 1.002 (1.000–1.003) and 0.80 (0.21–0.84) for treatment with medium alone and estrogen respectively. The median MFI expression of ZO-1 in Caco-2 cells assessed by microscopy was 0.97 (0.77–1.14) and 1.16 (0.76–1.37) for treatment with estrogen and medium alone respectively (P = 0.01). The median luciferase activity of NK-κB in Caco-2 cells assessed by NK-κB reporter activity assay was 0.58 (0.53–0.60) and 0.98 (0.87–1.05) for treatment with estrogen and medium alone respectively (P = 0.002). Conclusion Estrogen inhibits the ZO-1 expression and NF-κB activity in human gut mucosa.

Abstract A22: Association of breast cancer risk and concentrations of tissue estrogens to single strand breaks in DNA

Abstract Introduction: Single-strand breaks (SSB) in DNA are discontinuities in one strand of the DNA and are usually accompanied by loss of a single base and by damaged 5- or 3-termini at the site of the break. If not repaired rapidly or appropriately, chromosomal SSBs pose a serious threat to genetic stability and cancer development. We hypothesize that if the presence of single strand DNA breaks can be quantified directly, it will provide a means of detecting a process that puts cells at high risk of developing cancer. The association between a quantitative measure of SSB and other measures of breast cancer risk was determined. Subjects: 206 postmenopausal and 99 premenopausal, healthy women with intact, healthy bilateral breasts, without implants or history of radiation, willing to undergo a random fine-needle aspiration (rFNA) of the breast within 3.5 months of a normal mammogram were recruited to the study. Exclusions: use of tamoxifen, raloxifene, or aromatase inhibitor within 2 years of participation or oral contraceptives or other hormone treatments within 3 months of study enrollment. Participants completed personal and medical history questionnaires. Blood for hormone levels and random fine needle aspirates of the breast (rFNA) were collected following a breast exam. Methods: rFNA of the breast of women at unspecified risks for breast cancer were analyzed for SSB by a nick translation procedure. SSB levels digital two-dimensional breast density of the entire breast (PBD), mRNA of genes associated with DNA damage, and breast steroid concentrations by a LC/MS/MS procedure. Results: Based on the cpm in the purified sample, the specific activity of the 3H-dCTP, and quantity of DNA in the sample, the incorporation of 3H-dCTP ranged from 0.03 to 8.59 pmol/µg DNA. The β-coefficients for the relationships between SSB and measures of breast cancer risk were determined by a multiple regression procedure. PBD adjusted for age was associated with SSB in postmenopausal women (P = 0.007) but was not associated with SSB in premenopausal women. Further adjustment for BMI reduced the PBD relationship to SSB by 35% but adjustment for BMI. APEX1 was not significantly associated with SSB. XRCC1 mRNA was negatively associated with SSB in premenopausal women (p = 0.016), and was not altered by adjustment for age. The antioxidant functions NRF-1 and SOD2 were both significantly negatively associated with SSBs, P = 0.001 and 0.045, respectively, and both were decreased by less than 5% after adjustment for age. Breast tissue concentrations of 4-hydroxyestradiol exceeded those of estradiol, were correlated with tissue estradiol, and were significantly (P = 0.011) negatively related to SSB levels. Breast tissue concentrations of estradiol, estrone, 4-hydroxyestrone, and androstenedione were not significantly related to SSB. Conclusions: The most likely mechanism by which 4-OHE2 or 2-OHE1 could protect against formation of SSBs in the breast is by their antioxidative properties. 4-OHE2 and other catechol estrogens are capable of undergoing redox reactions, cycling between the catechol structure, semiquinone radical, and o-quinone. They are also capable of forming metal complexes, sequestering metals, preventing them from undergoing redox reactions. In addition, 4-OHE2 has a binding affinity relative to that of estradiol of 1.51; that of 2-OHE1 is 1.02 Therefore, the catechol estrogens have activity similar to or greater than that of estradiol in terms of activation of NRF-1 and Mn SOD, both of which were elevated in association with lower SSB levels. We conclude that SSBs measured by the nick translation procedure are associated with, but not redundant with, measures of breast cancer risk and with deficiencies of both DNA damage responses and antioxidant mechanisms. Concentrations of 4-hydroxyestradiol in breast tissue may serve an antioxidant function and may be protective. Citation Format: Mathavi Sahadevan, Oukseub Lee, Miguel Muzzio, Belinda Phan, Lisa Jacobs, Nagi Khouri, Jun Wang, Hong Hu, Vered Stearns, Robert T. Chatterton. Association of breast cancer risk and concentrations of tissue estrogens to single strand breaks in DNA. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A22.

Age- and menopause-related differences in subcutaneous adipose tissue estrogen receptor mRNA expression

ObjectivesChanges in estrogen receptor (ER) expression likely underlie differential metabolic effects of estrogen in pre- and postmenopausal women. The aim of the current study was to determine whether ER gene expression in abdominal and femoral subcutaneous adipose tissue (SAT) was associated with age, menopause, or regional adiposity. MethodsWe studied pre- and post-menopausal (n=23 and 22, respectively; age 35–65y) normal weight (mean±SD; BMI 23.7±2.5kg/m2) women with similar total fat mass. Abdominal and femoral SAT ERα (ESR1) and ERβ (ESR2) mRNA expression was determined by qPCR. ResultsTotal fat mass did not differ between pre- and postmenopausal women (22.7±5.3vs. 21.7±5.3kg). Compared to premenopausal women, ESR1 and the ratio of ESR1 to ESR2 were lower (p≤0.05) in postmenopausal abdominal and femoral SAT. ESR1 and ESR1:ESR2 were inversely associated with age in abdominal SAT (r=−0.380 and r=−0.463, respectively; p<0.05) and femoral SAT (r=−0.353 and r=−0.472, respectively; p<0.05). ESR2 was not related to age or menopause. The inverse association between ESR1 and age persisted after adjusting for trunk fat mass, estradiol, or leptin. ConclusionAmong healthy pre- and postmenopausal women, increased age was associated with a decreased balance of ERα to ERβ in abdominal and femoral subcutaneous adipose tissue.

Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells

GPER1, also known as GPR30, is a novel seven-transmembrane G-protein coupled estrogen receptor that mediates both short-term (non-genomic) and long-term (genomic) effects of estrogen in target cells and tissues. A substantial body of work over the last two decades has highlighted its therapeutic or prognostic utility. However, the clinical data on the expression of GPER1 in breast tissue is ambiguous. Analysis of TCGA RNAseq data revealed significantly lower mean expression of GPER1 mRNA in primary breast tumors compared to that in normal breast tissues. This provides support to the tumor suppressor role for GPER1. However, the mechanisms underlying the reduced expression are not completely understood. We analyzed the expression levels of GPER1 mRNA variants in MCF-7 and MDA-MB-231 cells by RT-PCR, and the methylation status of two CpG islands in the GPER1 locus by modified COBRA assays and bisulfite sequencing. Our results show that MCF-7 cells express higher levels of GPER1 mRNA variants compared to MDA-MB-231 cells. Modified COBRA assays revealed differential methylation in the upstream CpG island (upCpGi) that overlaps with the first exon of two GPER1 variants (GPER1v2 and v3) but not in the downstream CpG island (dnCpGi) that overlaps with the coding region common to all variants. Bisulfite sequencing results showed that the core upCpGi was hypo-methylated in both MCF-7 and MDA-MB-231 cells. However, eight CpGs in the 3′ end of the upCpGi were hyper-methylated in MDA-MB-231 cells. 5-Azacytidine, a DNA methyltransferase inhibitor, induced the expression levels of GPER1 mRNA variants in MDA-MB-231 cells. Expression-methylation correlation analysis of TCGA breast cancer data revealed that methylation of CpGs in the regions flanking the upCpGi significantly correlated negatively with GPER1 mRNA expression. Taken together, our results demonstrate the role of DNA methylation in GPER1 repression, implicate the flanking regions (shore) of the upCpGi, and suggest a potential mechanism of GPER1 silencing in breast tumors.

Effect of β-agonist on the dexamethasone-induced expression of aromatase by the human monocyte cells.

Emerging evidence suggests that sex steroids are important for human skin health. In particular, estrogen improves skin thickness, elasticity and moisture of older women. The major source of circulating estrogen is the ovary; however, local estrogen synthesis and secretion have important roles in, for example, bone metabolism and breast cancer development. We hypothesized that infiltrated peripheral monocytes are one of the sources of estrogen in skin tissues. We also hypothesized that, during atopic dermatitis under stress, a decline in the hypothalamus–pituitary–adrenal axis (HPA) and facilitation of the (hypothalamus)–sympathetic–adrenomedullary system (SAM) attenuates estrogen secretion from monocytes. Based on this hypothesis, we tested aromatase expression in the human peripheral monocyte-derived cell line THP-1 in response to the synthetic glucocorticoid dexamethasone (Dex), the synthetic β-agonist isoproterenol (Iso) and the β-antagonist propranolol (Pro). Dex mimics glucocorticoid secreted during excitation of the HPA, and Iso mimics catecholamine secreted during excitation of the SAM. We found that aromatase activity and the CYP19A1 gene transcript were both upregulated in THP-1 cells in the presence of Dex. Addition of Iso induced their downregulation and further addition of Pro rescued aromatase expression. These results may suggest that attenuation of estrogen secretion from peripheral monocytes could be a part of the pathology of stress-caused deterioration of atopic dermatitis. Further examination using an in vitro human skin model including THP-1 cells might be a valuable tool for investigating the therapeutic efficacy and mechanism of estrogen treatment for skin health.

Estrogen Biosynthesis and Its Regulation in Endometriosis

Endometriosis is a common benign gynecological disorder with an enigmatic etiology and pathogenesis. It affects approximately 10% women of reproductive age. Although its etiology and pathogenesis remain poorly understood, it is characterized by the elevated local production of estrogen in the endometriotic tissues. In this paper, we review the mechanisms of estrogen biosynthesis and its regulation in endometriosis.