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Objectives: Inhibition of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, the rate rate-determining enzyme for the biogenesis of cholesterol is known to show antineoplastic effects. Therefore, this study investigates the in-silico HMG-CoA reductase (HMGCR)-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin., Methods: Carotenoids from S. mombin leaves were characterized with the aid of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The characterized phytochemicals were obtained from PubChem. They were docked into the orthosteric site of human HMGCR (Protein Data Bank code 1HW8) using AutoDock 4.0 suites. DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer was treated with the carotenoids extract from S. mombin (100 mg/kg and 200 mg/kg doses) to assess its anti-lipidaemic cum anticancer effects., Key Findings: Carotenoids from S. mombin; beta-carotene-15,15'-epoxide, astaxanthin and 7,7',8,8'-tetrahydro-β-β-carotene demonstrate HMGCR inhibition. They form hydrophobic interactions with key residues within the catalytic domain of HMGCR. The carotenoids extract exhibits anti-lipidaemic/anticancer effects, lowering serum triglyceride, LDL and cholesterol concentration. It increases HDL concentration and downregulates the expression of HMGR, AFP, CEACAM-3, BRCA-1 and HIF-1 mRNAs., Conclusion: Carotenoids from S. mombin demonstrate HMG-CoA reductase (HMGCR) inhibition, anti-lipidaemic, and anticancer effects. The inhibition of HMGCR by the carotenoids extract further poses it as a potential anti-hypercholesterolaemia compounds., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Breast development is important for most trans women. An important limitation of current breast development measurement methods is that these do not allow for 3D volume analyses., Objectives: To examine breast development and change in anthropometry during the first 3 years of gender-affirming hormone treatment using 3D imaging. Associations with clinical or laboratory parameters and satisfaction with the gained breast development were also studied., Design: Prospective cohort study., Setting: Specialized tertiary gender identity clinic in Amsterdam, the Netherlands., Participants: Participants were 69 adult trans women with a median age of 26 years (interquartile range, 21-38)., Interventions: Gender-affirming hormone treatment., Main Outcome Measures: Volumetric and anthropometric breast development and satisfaction., Results: Breast volume increased by 72 cc (95% confidence interval [CI], 48-97) to 100 cc (standard deviation 48). This resulted in a cup-size

Background: Although physical activity has been consistently associated with reduced breast cancer mortality, evidence is largely based on data collected at one occasion. We examined how pre- and postdiagnosis physical activity was associated with survival outcomes in high-risk breast cancer patients., Methods: Included were 1340 patients enrolled in the Diet, Exercise, Lifestyle and Cancer Prognosis (DELCaP) Study, a prospective study of lifestyle and prognosis ancillary to a SWOG clinical trial (S0221). Activity before diagnosis, during treatment, and at 1- and 2-year intervals after enrollment was collected. Patients were categorized according to the Physical Activity Guidelines for Americans as meeting the minimum guidelines (yes/no) and incrementally as inactive, low active, moderately active (meeting the guidelines), or high active., Results: In joint-exposure analyses, patients meeting the guidelines before and 1 year after diagnosis experienced statistically significant reductions in hazards of recurrence (hazard ratio [HR] = 0.59, 95% confidence interval [CI] = 0.42 to 0.82) and mortality (HR = 0.51, 95% CI = 0.34-0.77); associations were stronger at 2-year follow-up for recurrence (HR = 0.45, 95% CI = 0.31 to 0.65) and mortality (HR = 0.32, 95% CI = 0.19 to 0.52). In time-dependent analyses, factoring in activity from all time points, we observed striking associations with mortality for low- (HR = 0.41, 95% CI = 0.24 to 0.68), moderate- (HR = 0.42, 95% CI = 0.23 to 0.76), and high-active patients (HR = 0.31, 95% CI = 0.18 to 0.53)., Conclusions: Meeting the minimum guidelines for physical activity both before diagnosis and after treatment appears to be associated with statistically significantly reduced hazards of recurrence and mortality among breast cancer patients. When considering activity from all time points, including during treatment, lower volumes of regular activity were associated with similar overall survival advantages as meeting and exceeding the guidelines., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Context: Previous case reports associated prepubertal gynecomastia with lavender-containing fragrances, but there appear to be no reports of premature thelarche., Objective: To add to a case series about lavender-fragranced product use and breast growth in children and to measure endocrine-disrupting chemical activity of essential oil components., Design, Setting, and Patients: Patients experiencing premature thelarche or prepubertal gynecomastia with continuous exposure to lavender-fragranced products were evaluated in the pediatric endocrinology departments of two institutions. Mechanistic in vitro experiments using eight components of lavender and other essential oils were performed at National Institute of Environmental Health Sciences., Main Outcome Measures: Case reports and in vitro estrogen and androgen receptor gene expression activities in human cell lines with essential oils., Results: Three prepubertal girls and one boy with clinical evidence of estrogenic action and a history of continuous exposure to lavender-containing fragrances were studied. Breast growth dissipated in all patients with discontinuation of the fragranced products. Some of the components tested elicited estrogenic and antiandrogenic properties of varying degrees., Conclusion: We report cases of premature thelarche that resolved upon cessation of lavender-containing fragrance exposure commonly used in Hispanic communities. The precise developmental basis for such conditions could be multifactorial. In vitro demonstration of estrogenic and antiandrogenic properties of essential oil components suggests essential oils in these cases could be considered a possible source and supports a possible link with idiopathic prepubertal breast development. Whether the level of lavender oil estrogenic potency is sufficient to cause these effects is unknown., (Copyright © 2019 Endocrine Society.)

Context: Medroxyprogesterone acetate (MPA) is a widely used progestin in feminizing hormone therapy. However, the side effects and hormonal changes elicited by this drug have never been investigated in the transgender population., Objective: We evaluated the incidence of self-reported effects among transwomen using MPA and this drug's impact on hormonal and metabolic parameters., Design, Setting, and Participants: We retrospectively collected data from 290 follow-up visits (FUVs) of transwomen treated at Rhode Island Hospital from January 2011 to July 2018 (mean duration of therapy 3.4 ± 1.7 years). FUVs followed regimens of estradiol (E) and spironolactone, with MPA (n = 102) or without MPA (n = 188)., Main Outcome Measures: We assessed the incidence of self-reported effects after MPA treatment. We also compared blood levels of E, testosterone, and various laboratory parameters between MPA and non-MPA groups., Results: Mean weighted E level was 211 ± 57 pg/mL after MPA treatment and 210 ± 31 pg/mL otherwise; this difference was nonsignificant [t(274) = 0.143, P = 0.886]. Mean weighted testosterone level was 79 ± 18 ng/dL after MPA treatment and 215 ± 29 ng/dL otherwise; testosterone levels were significantly lower in the MPA group [t(122) = 32.4, P < 0.001]. There were minimal changes in other laboratory parameters. Of 39 patients receiving MPA, 26 reported improved breast development and 11 reported decreased facial hair. Five patients experienced mood swings on MPA., Conclusions: In our cohort of transwomen, we found minimal side effects, unchanged E levels, and a decline in testosterone associated with MPA, outcomes consistent with feminization. Prospective studies are needed to confirm our findings., (Copyright © 2019 Endocrine Society.)

As a continuous source of hormonal stimulation, environmentally ubiquitous estrogenic chemicals, ie, xenoestrogens (XEs), are a potential risk factor for breast carcinogenesis. Given their wide distribution in the environment and the fact that bisphenol-A (BPA), methylparaben (MP), and perfluorooctanoic acid (PFOA) are uniformly detected in unselected body fluid samples, it must be assumed that humans are simultaneously exposed to these chemicals almost daily. We studied the effects of a ternary mixture of BPA, MP, and PFOA on benign breast epithelial cells at the range of concentrations observed for single chemicals in human samples. Measurements of exposure impact relevant to the breast were based on endpoints associated with "hallmarks" of cancer and "key characteristics" of carcinogens. These included modulation of total estrogen receptor (ER)α, phosphorylated ERα (pERα), total ERβ, S-phase induction, and apoptotic evasion. Data from live cell measurements were fit to a log-linear dose-response model. Concentration-dependent reduction of ERβ and apoptosis evasion was observed concurrently with the induction of ERα, pERα, and S-phase fraction, and an increased rate of cell proliferation. Beyond additive effects predicted by the sum of individual test XEs, mixture treatment demonstrated synergism for the ERβ and apoptosis suppression phenotypes (p > .001). Nonmalignant breast cells were more sensitive than commonly used breast cancer lines to XE treatment in 3 of 5 endpoints. All observations were validated with cells isolated from the normal breast tissue of 14 individuals. At relatively low concentrations, a chemical mixture has striking effects on normal cell function that are missed by evaluation of single components.

Developmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cells have not been measured. Here, we quantified the effects of cadmium exposure on reduction mammoplasty patient-derived breast stem cell proliferation and differentiation. Using the mammosphere assay and organoid formation in 3D hydrogels, we tested 2 physiologically relevant doses of cadmium, 0.25 and 2.5 µM, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a hypoxia-inducible factor (HIF)-1α activity reporter line and pharmaceutical inhibition of HIF-1α in organoid formation assays. 2.5 µM cadmium reduced primary mammosphere formation and branching structure organoid formation rates by 33% and 87%, respectively. Despite no changes in mammosphere formation, 0.25 µM cadmium inhibited branching organoid formation in hydrogels by 73%. RNA-seq revealed cadmium downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium downregulated HIF-1α target genes including LOXL2, ZEB1, and VIM. Cadmium significantly inhibited HIF-1α activity in a luciferase assay, and the HIF-1α inhibitor acriflavine ablated mammosphere and organoid formation. These findings show that cadmium, at doses relevant to human exposure, inhibited human mammary stem cell proliferation and differentiation, potentially through disruption of HIF-1α activity.

Purpose: Chemicals with hormonelike activity, such as estrogenic isoflavones, may perturb human development. Infants exclusively fed soy-based formula are highly exposed to isoflavones, but their physiologic responses remain uncharacterized. Estrogen-responsive postnatal development was compared in infants exclusively fed soy formula, cow-milk formula, and breast milk., Methods: We enrolled 410 infants born in Philadelphia-area hospitals between 2010 and 2014; 283 were exclusively fed soy formula (n = 102), cow-milk formula (n = 111), or breast milk (n = 70) throughout the study (birth to 28 or 36 weeks for boys and girls, respectively). We repeatedly measured maturation index (MI) in vaginal and urethral epithelial cells using standard cytological methods, uterine volume and breast-bud diameter using ultrasound, and serum estradiol and follicle-stimulating hormone levels. We estimated MI, organ-growth, and hormone trajectories by diet using mixed-effects regression splines., Results: Maternal demographics did not differ between cow-milk-fed and soy-fed infants but did differ between formula-fed and breastfed infants. Vaginal-cell MI trended higher (P = 0.01) and uterine volume decreased more slowly (P = 0.01) in soy-fed girls compared with cow-milk-fed girls; however, their trajectories of breast-bud diameter and hormone concentrations did not differ. We observed no significant differences between boys fed cow-milk vs soy formula; estradiol was not detectable. Breastfed infants differed from soy-formula-fed infants in vaginal-cell MI, uterine volume, and girls' estradiol and boys' breast-bud diameter trajectories., Conclusions: Relative to girls fed cow-milk formula, those fed soy formula demonstrated tissue- and organ-level developmental trajectories consistent with response to exogenous estrogen exposure. Studies are needed to further evaluate the effects of soy on child development.

Context: Breast development is a key feature of feminization and therefore important to transwomen (male-to-female transgender persons). It is not exactly known when breast development starts after initiating cross-sex hormone therapy (CHT) and how much growth may be expected., Objective: To investigate breast development in transwomen during their first year of CHT and whether clinical or laboratory parameters predict breast development., Design: This study was performed as part of the European Network for the Investigation of Gender Incongruence, which is a prospective multicenter cohort study., Setting: Gender clinics in Amsterdam, Ghent, and Florence., Participants: Transwomen who completed the first year of CHT (n = 229)., Intervention: CHT., Main Outcome Measures: Breast development in centimeter and cup size., Results: The median age of the included transwomen was 28 years (range, 18 to 69). Mean breast-chest difference increased to 7.9 ± 3.1 cm after 1 year of CHT, mainly resulting in less than an AAA cup size (48.7%). Main breast development occurred in the first 6 months of therapy. Serum estradiol levels did not predict breast development after 1 year of CHT (first quartile, 3.6 cm [95% confidence interval (CI), 2.7 to 4.5], second quartile, 3.2 cm [95% CI, 2.3 to 4.2], third quartile, 4.4 cm [95% CI, 3.5 to 5.3], and fourth quartile, 3.6 cm [95% CI, 2.7 to 4.5])., Conclusion: This study shows that, after 1 year of CHT, breast development is modest and occurs primarily in the first 6 months. No clinical or laboratory parameters were found that predict breast development., (Copyright © 2017 Endocrine Society)

Hyperglycemia is an important factor for chemoresistance of breast cancer patients with diabetes. In the present study, a novel selenadiazole derivative has been evaluated and found to be able to antagonize the doxorubicin (DOX) resistance of MCF-7 cells under simulated diabetes conditions. Hyperglycemia promotes the proliferation, invasion and migration of MCF-7 cells through activation of ERK and AKT pathways, which could be inhibited by the synthetic selenadiazole derivative. The antitumor effects of the selenadiazole derivative were attributed to its ability to activate AMPK pathways. Furthermore, the high lipophilicity (log P = 1.9) of the synthetic selenadiazole derivative facilitated its uptake by cancer cells and subsequently potentiated the cellular uptake of DOX, leading to a strong enhancment of the antiproliferative activity of DOX on MCF-7 cells by induction of apoptosis. The apoptosis was initiated by the ROS overproduction induced by the cooperation of the selenadiazole derivative and DOX. The excessive ROS then caused damage to DNA, which upregulated the expression of proapoptosis Bcl-2 family proteins and led to fragmentation of mitochondria, which finally caused apoptosis of the cancer cells. Taken together, this study provides a rational strategy for using selenadiazole derivatives to overcome hyperglycemia-induced drug resistance in breast cancer by activation of AMPK-mediated pathways.

Background: Biomarkers may play an important role in identifying patients at risk for cancer therapy cardiotoxicity. Our objectives were to define the patterns of change in biomarkers with cancer therapy and their associations with cardiotoxicity., Methods: In a multicenter cohort of 78 breast cancer patients undergoing doxorubicin and trastuzumab therapy, 8 biomarkers were evaluated at baseline and every 3 months over a maximum follow-up of 15 months. These biomarkers, hypothesized to be mechanistically relevant to cardiotoxicity, included high-sensitivity cardiac troponin I (hs-cTnI), high-sensitivity C-reactive protein (hsCRP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), growth differentiation factor 15 (GDF-15), myeloperoxidase (MPO), placental growth factor (PlGF), soluble fms-like tyrosine kinase receptor-1 (sFlt-1), and galectin 3 (gal-3). We determined if biomarker increases were associated with cardiotoxicity at the same visit and the subsequent visit over the entire course of therapy. Cardiotoxicity was defined by the Cardiac Review and Evaluation Criteria; alternative definitions were also considered., Results: Across the entire cohort, all biomarkers except NT-proBNP and gal-3 demonstrated increases by 3 months; these increases persisted for GDF-15, PlGF, and hs-cTnI at 15 months. Increases in MPO, PlGF, and GDF-15 were associated with cardiotoxicity at the same visit [MPO hazard ratio 1.38 (95% CI 1.10-1.71), P = 0.02; PlGF 3.78 (1.30-11.0), P = 0.047; GDF-15 1.71 (1.15-2.55), P = 0.01] and the subsequent visit. MPO was robust to alternative outcome definitions., Conclusions: Increases in MPO are associated with cardiotoxicity over the entire course of doxorubicin and trastuzumab therapy. Assessment with PlGF and GDF-15 may also be of value. These findings motivate validation studies in additional cohorts., (© 2015 American Association for Clinical Chemistry.)

The increasing use of chemotherapy before surgery has affected a number of local-regional treatment decisions including surgical and radiation management of the breast, management of axillary lymph nodes, and the indications for postmastectomy radiation. In this monograph, we will focus on surgical and radiation management as components of breast conservation therapy. The early randomized trials that compared neoadjuvant to adjuvant chemotherapy in breast cancer demonstrated that rates of breast conservation can be increased when chemotherapy is sequenced first. This was a direct consequence of high response rates seen with neoadjuvant treatment, which permitted downstaging of a large primary tumor to a volume that permitted breast-conserving surgery. Some initial studies found higher rates of breast recurrences with this approach but over time, with improved multidisciplinary coordination and proper patient selection, rates of breast recurrences have improved to the excellent levels achieved when surgery is performed first. New clinical trials are also ongoing to define the role of sentinel lymph node surgery and regional lymph node radiation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The use of imaging in the arena of primary treatment for breast cancer is gaining importance as a technique for assessing response to chemotherapy as well as assessing the underlying tumor biology. Both mammography and ultrasound have traditionally been used, in addition to clinical evaluation, to evaluate response to treatment although they have shed little light on the underlying biological processes. Functional magnetic resonance imaging techniques have the ability to assess response to treatments in addition to providing valuable information on changes in tumor perfusion, vascular permeability, oxygenation, cellularity, proliferation, and metabolism both at baseline and after treatment. This noninvasive method of evaluating cellular function is of importance both as endpoints for clinical trials and to our understanding of the biological mechanisms of cancer., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: In trans women (male-to-female transsexual persons), cross-sex hormone therapy is administered to induce feminization. Breast development is an important part of feminization for most trans women., Aim: The aim of this study is to assess the effect of cross-sex hormone therapy on breast development in adult trans women. Additionally, we aimed to investigate the benefit or harm of administration of progestogens on breast development., Methods: A review of the literature in Embase, Medline, The Cochrane Library, PsycINFO databases, PubMed, and Web of Knowledge until January 2014., Main Outcome Measures: Effects of cross-sex hormone therapy and progestogens on breast development in trans women., Results: Only few studies with low quality of evidence addressed these topics. The available evidence suggests that breast development is insufficient for the majority of trans women and that type and dosage of hormonal therapy seem not to have an important role on final breast size., Conclusions: Our knowledge concerning the natural history and effects of different cross-sex hormone therapies on breast development in trans women is extremely sparse and based on low quality of evidence. Current evidence does not provide evidence that progestogens enhance breast development in trans women. Neither do they prove the absence of such an effect. This prevents us from drawing any firm conclusion at this moment and demonstrates the need for further research to clarify these important clinical questions., (© 2014 International Society for Sexual Medicine.)

Objective: Short stature is a prominent feature of Turner syndrome (TS), which is partially overcome by GH treatment. We have previously reported the results of a trial on the effect of oxandrolone (Ox) in girls with TS. Ox in a dose of 0.03 mg/kg per day (Ox 0.03) significantly increased adult height gain, whereas Ox mg/kg per day (0.06) did not, at the cost of deceleration of breast development and mild virilization. The aim of this follow-up study in adult participants of the pediatric trial was to investigate the long-term effects of previous Ox treatment., Design and Methods: During the previous randomized controlled trial, 133 girls were treated with GH combined with placebo (Pl), Ox 0.03, or Ox 0.06 from 8 years of age and estrogen from 12 years. Sixty-eight women (Pl, n=23; Ox 0.03, n=27; and Ox 0.06, n=18) participated in the double-blind follow-up study (mean age, 24.0 years; mean time since stopping GH, 8.7 years; and mean time of Ox/Pl use, 4.9 years). We assessed height, body proportions, breast size, virilization, and body composition., Results: Height gain (final minus predicted adult height) was maintained at follow-up (Ox 0.03 10.2±4.9 cm, Ox 0.06 9.7±4.4 cm vs Pl 8.0±4.6 cm). Breast size, Tanner breast stage, and body composition were not different between groups. Ox-treated women reported more subjective virilization and had a lower voice frequency., Conclusion: Ox 0.03 mg/kg per day has a beneficial effect on adult height gain in TS patients. Despite previously reported deceleration of breast development during Ox 0.03 treatment, adult breast size is not affected. Mild virilization persists in only a small minority of patients. The long-term evaluation indicates that Ox 0.03 treatment is effective and safe.

Introduction: Sex steroid exposure increases the risk of breast cancer by unclear mechanisms. Diet modifications may be one breast cancer prevention strategy. The proinflammatory cytokine family of IL-1 is implicated in cancer progression. IL-1Ra is an endogenous inhibitor of the proinflammatory IL-1α and IL-1β., Objective: The objective of this study was to elucidate whether estrogen, tamoxifen, and/or diet modification altered IL-1 levels in normal human breast tissue., Design and Methods: Microdialysis was performed in healthy women under various hormone exposures, tamoxifen therapy, and diet modifications and in breast cancers of women before surgery. Breast tissue biopsies from reduction mammoplasties were cultured., Results: We show a significant positive correlation between estradiol and in vivo levels of IL-1β in breast tissue and abdominal sc fat, whereas IL-1Ra exhibited a significant negative correlation with estradiol in breast tissue. Tamoxifen or a dietary addition of 25 g flaxseed per day resulted in significantly increased levels of IL-1Ra in the breast. These results were confirmed in ex vivo culture of breast biopsies. Immunohistochemistry of the biopsies did not reveal any changes in cellular content of the IL-1s, suggesting that mainly the secreted levels were affected. In breast cancer patients, intratumoral levels of IL-1β were significantly higher compared with normal adjacent breast tissue., Conclusion: IL-1 may be under the control of estrogen in vivo and may be attenuated by antiestrogen therapy and diet modifications. The increased IL-1β in breast cancers of women strongly suggests IL-1 as a potential therapeutic target in breast cancer treatment and prevention.

Background: Antiprogestins are of growing interest for the development of new treatments in the gynecological field. Ulipristal acetate (UPA) is a progesterone receptor (PR) modulator considered for long-term administration in contraception and is currently being registered for the treatment of uterine fibroids. In light of the influences of hormonal dysfunction in breast pathologies, the secondary consequences of chronic UPA therapy need to be established. The aim of this study was to determine UPA actions mediated by PR and glucocorticoid receptor (GR) in normal and transformed breast., Methods: UPA, progesterone (P) and dexamethasone (DEX) effects were observed on PR and GR responsive genes and on proliferation and apoptosis of normal human breast epithelial (HBE) and breast cancer cells. Human normal breast tissue samples were xenografted in athymic mice and treated with estradiol (E2), or E2 + P, or E2 + P + UPA., Results: Analysis of PR and GR reporter gene transactivation and their respective endogenous target genes indicated that UPA exerted anti-progestational and anti-glucocorticoid activity in both types of cells with a more pronounced effect in cancer cells. When combined with P or DEX, UPA limits the proliferation of HBE cells but increases growth in breast cancer cell lines. UPA administration had no impact on the mitotic index on xenografted human breast tissue exposed to gonadal hormones at similar concentrations to those present in normal women., Conclusions: Although further clinical trials are required to confirm that the results from our experimental models can be extrapolated to women treated with UPA, they suggest that such treatment would not be deleterious to normal breast tissue at least for a cycle (28 days) of continuous administration.

Background: The data concerning the effects and safety of androgen in human breast tissue are conflicting., Objective: Our aim was to analyze the effects of androgens on normal human breast tissue (HBT)., Approach: We cultured explants of HBT (obtained from reduction mammoplasty operations of postmenopausal women) with or without testosterone (T) and 5α-dihydrotestosterone (DHT) or in combination with 17β-estradiol (E(2)) for 7 and 14 d to study the effects of androgens on proliferation, apoptosis, target gene expression, and steroid receptors. The androgen receptor (AR) and estrogen receptor (ER) dependences of the effects were studied with the antihormones bicalutamide and fulvestrant, respectively., Results: The hormone responsiveness of cultured breast tissue was assessed by assaying apolipoprotein-D and prostate-specific antigen expression increased by androgens and amphiregulin and trefoil factor-1 expression induced by E(2) treatment. T and DHT reduced proliferation and increased apoptosis in breast epithelium, the effects of which were reversed by bicalutamide. In combination with E(2), they suppressed E(2)-stimulated proliferation and cell survival. DHT also inhibited basal (P < 0.05) and E(2)-induced expression of cyclin-D1 mRNA (P < 0.05). Immunohistochemistry showed that T (P < 0.05) and DHT (P < 0.05) increased the relative number of AR-positive cells, whereas ERα-positive (P < 0.001) cell numbers were strongly decreased. The percentage of ERβ-positive cells remained unchanged. E(2) treatment increased ERα-positive (P < 0.01) cells, whereas AR- (P < 0.05) and ERβ-expressing (P < 0.001) cells diminished. These effects were repressed in combination cultures of E(2) with T and DHT., Conclusion: T and DHT inhibited proliferation and increased apoptosis in the epithelium of cultured normal HBT and opposed E(2)-stimulated proliferation and cell survival in an AR-dependent manner. These effects were associated with changes in the proportions of ERα- and AR-positive epithelial cells.

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

Background: Mammographic breast density is a strong risk factor for breast cancer. Tamoxifen, which reduces the risk of breast cancer in women at high risk, also reduces mammographic breast density. However, it is not known if tamoxifen-induced reductions in breast density can be used to identify women who will benefit the most from prophylactic treatment with this drug., Methods: We conducted a nested case-control study within the first International Breast Cancer Intervention Study, a randomized prevention trial of tamoxifen vs placebo. Mammographic breast density was assessed visually and expressed as a percentage of the total breast area in 5% increments. Case subjects were 123 women diagnosed with breast cancer at or after their first follow-up mammogram, which took place 12-18 months after trial entry, and control subjects were 942 women without breast cancer. Multivariable logistic regression was used to adjust for other risk factors. All statistical tests were two-sided., Results: In the tamoxifen arm, 46% of women had a 10% or greater reduction in breast density at their 12- to 18-month mammogram. Compared with all women in the placebo group, women in the tamoxifen group who experienced a 10% or greater reduction in breast density had 63% reduction in breast cancer risk (odds ratio = 0.37, 95% confidence interval = 0.20 to 0.69, P = .002), whereas those who took tamoxifen but experienced less than a 10% reduction in breast density had no risk reduction (odds ratio = 1.13, 95% confidence interval = 0.72 to 1.77, P = .60). In the placebo arm, there was no statistically significant difference in breast cancer risk between subjects who experienced less than a 10% reduction in mammographic density and subjects who experienced a greater reduction., Conclusion: The 12- to 18-month change in mammographic breast density is an excellent predictor of response to tamoxifen in the preventive setting.

Introduction: The safety of long-term physiological doses of testosterone (T) therapy in women with sexual dysfunction is a contentious issue, in part, because of fear of adverse effects, such as breast cancer, vascular disease, and excessive virilization. This unsubstantiated fear has hampered progress in treating women with sexual dysfunction using T therapy in physiological doses to achieve circulating levels in the normal range., Aim: To examine evidence derived from studies in female-to-male transsexuals (FMT) treated with supraphysiological (pharmacological) doses of T for long periods of time with no apparent major adverse effects., Methods: A comprehensive literature search of relevant articles published between 1980 and 2010 pertaining to the topic of T in FMTs was performed using PubMed. The following key words were used: female-to-male transsexuals; testosterone; virilization; gender re-assignment; and androgen therapy in women. Relevant articles were retrieved, reviewed, and the information was analyzed and evaluated for study methodology and major findings., Main Outcome Measures: Data from peer-reviewed publications were critically analyzed and the information was summarized., Results: The data from the studies reported in the literature to date strongly suggest that treatment of FMTs with supra-physiological doses of T had minimal adverse effects. No increase in mortality, breast cancer, vascular disease, or other major health problems were reported., Conclusions: No significant serious adverse effects were reported in FMTs treated with pharmacological doses of T. In light of the findings with supraphysiological doses of T, we suggest that treatment with T at doses producing physiological levels in women with sexual dysfunction are expected to produce limited and minimal adverse effects., (© 2010 International Society for Sexual Medicine.)

Introduction: The most common treatment regimen in female-to-male transsexuals is administration of short-acting testosterone esters intramuscularly every 2 weeks., Aim: The aim of this study was to evaluate the effect of long-acting intramuscular testosterone undecanoate on body composition and bone mineral density during cross-sex hormone therapy in female-to-male transsexuals., Methods: Forty-five female-to-male transsexuals (FtMs) were treated with injections of testosterone undecanoate 1,000 mg intramuscularly every 12 weeks over 24 months., Main Outcome Measures: Body composition, bone mineral density, hormone parameters, and lipids were compared after 12 months and after 24 months with baseline values. Sonographic findings in the ovaries and endometrium, clinical and adverse effects during the study period were recorded., Results: There was a significant increase in lean mass in the FtMs during the study period in comparison with baseline values, whereas no change in BMI, fat mass, and bone mineral density was observed. There was a significant decline in gonadotropins, estradiol, dehydroepiandrosterone sulphate, sex hormone-binding globulin, and high-density lipoprotein, while testosterone and triglyceride levels increased significantly after 12 and 24 months. Ovaries remained unchanged and no noticeable endometrial pathology was observed. No mortality or morbidity was observed during the study period. We observed a cessation of menstrual bleeding, an increase in clitoral growth, libido, body and beard hair growth, deepened voices and decline in breast size. There was a significant increase in hemoglobin, hematocrit, glutamic-pyruvic transaminase, gamma-glutamyl transferase, and an increase in systolic blood pressure during the study period., Conclusions: There was an increase in lean mass during the study period in FtMs treated with testosterone undecanoate. Transsexual patients should be monitored for adverse effects on lipid profiles, blood pressure, and erythrocytosis during intramuscular testosterone undecanoate therapy., (© 2010 International Society for Sexual Medicine.)

Context: Use of aromatase inhibitors to suppress estrogen production is being actively investigated in a variety of experimental conditions in both females and males. Anastrozole (Arimidex) is a potent and selective reversible inhibitor of the aromatase enzyme in females., Objective: Our objective was to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of anastrozole in adolescent males with gynecomastia of less than 1 yr duration. The effect of anastrozole on breast size was also assessed as an exploratory aim., Design: We conducted a PK/PD open-label study., Setting: This clinical research center study was undertaken at pediatric academic centers., Patients: Forty-two boys with gynecomastia (mean age 13 +/- 1.8 yr; duration of gynecomastia 7.0 +/- 2.5 months; body mass index 28.3 +/- 5.9 kg/m(2)) were recruited., Interventions: Anastrozole, 1 mg, was given daily for 6 months., Main Outcomes: We assessed PK/PD of anastrozole after 14 d daily dosing and changes in breast size (exploratory aim) by manual tape measurements (area) and ultrasound (volume) after 6 months., Results: Anastrozole was rapidly absorbed orally (time to reach maximum concentration, 1 h) with a slow apparent clearance of 1.54 liters/h and a terminal half-life of 46.8 h. Testosterone/estradiol ratios increased significantly with concomitant increase in LH/FSH concentrations indicating aromatase blockade. There was a reduction in breast area (approximately 63%) and breast volume (approximately 57%) in the study group as compared with baseline (P = 0.004). The drug was well tolerated., Conclusions: Anastrozole is a potent aromatase inhibitor in adolescent males, with rapid absorption and slow elimination kinetics after oral dosing. Exploratory analysis of changes in breast size showed breast reduction in the cohort; this deserves further study.

It was the aim of this study to examine the association of the consumption of meat in general, meat prepared by different cooking methods and the dietary intake of heterocyclic aromatic amines (HCA) with the level of DNA adducts in the breast tissue of women undergoing reduction mammoplasty. Dietary intake of meat and HCA were assessed via questionnaire in 44 women undergoing reduction mammoplasty. DNA adduct analysis in breast tissue was performed by (32)P-postlabelling analysis. Spearman rank correlation coefficients (r) were calculated to examine the association of meat consumption and dietary HCA intake with tissue DNA adduct levels. A median DNA adduct level of 18.45 (interquartile range 12.81-25.65) per 10(9) nucleotides in breast tissue was observed; median HCA intake was 40.43 ng/day (interquartile range 19.55-102.33 ng/day). Total HCA intake (r = 0.33, P = 0.03), consumption of fried meat (r = 0.39, P = 0.01), beef (r = 0.32, P = 0.03) and processed meat (r = 0.51, P = 0.0004) were statistically significantly correlated with the level of DNA adducts in breast tissue. The detected DNA adducts could not be confirmed to be specific HCA-derived DNA adducts by comparison with external standards, using the (32)P-postlabelling assay. We observed strong correlations of dietary HCA intake and consumption of fried and processed meat with DNA adduct levels in breast tissue of 44 women. Since the detected DNA adducts were not necessarily specific only for HCA, it is possible that HCA intake is a surrogate of other genotoxic substances, such as polycyclic aromatic hydrocarbons, in meat prepared at high temperatures.

Background: Progestins as well as estrogens have a role in breast cell proliferation and the development of breast cancer. Here, the effect of mifepristone on cell proliferation in human breast tissue in vivo was studied in premenopausal women., Methods: A group of 30 women, scheduled for surgical treatment of leiomyomas, were randomized to either 50 mg mifepristone or placebo every other day, for 3 months. Fine needle aspiration biopsies were obtained at baseline and after 3 months. Immunocytochemical analysis of Ki-67 was performed to reflect breast epithelial cell proliferation. Samples from 14 women were included in the final analyses., Results: The Ki-67 index was significantly reduced after mifepristone treatment compared with baseline (P = 0.012). Furthermore, less individual variation in the Ki-67 index was seen in the mifepristone group. Treatment with mifepristone did not affect cortisol levels, whereas an increase in serum testosterone was noted. Breast symptoms like soreness and swelling were reduced, whereas the incidence of flushes increased., Conclusions: The ability of mifepristone to block breast epithelial cell proliferation in premenopausal women may prove beneficial when used for contraceptive purposes or for other gynaecological indications. Future studies should address a possible antiproliferative effect in the post-menopausal breast tissue during hormone replacement therapy. Our results implicate a possible protective effect of mifepristone on the breast epithelium. ClinicalTrials.gov NCT00579475.

Context: HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 also regulates differentiation of the adult reproductive tract and mammary gland in response to sex steroids., Objective: We recently identified two HOXA10 estrogen response elements (EREs). Here we demonstrate that estrogen-responsive HOXA10 expression is cell type specific., Design and Setting: We conducted an in vitro study at an academic medical center., Main Outcome Measure: Reporter assay, gel shift assays (electrophoretic mobility shift assay), and immunohistochemistry were done., Results: The HOXA10 EREs and a specificity protein 1 (Sp1) binding site differentially drive the cell-type-specific E2 response. In electrophoretic mobility shift assays, both estrogen receptor-alpha and -beta bound both EREs but not the Sp1 site. In reporter assays, both EREs and the Sp1 site demonstrated estrogen responsiveness and tissue specificity; transiently transfected uterine Ishikawa cells or breast MCF-7 cells showed differential responses to E2 treatment. Each response element (Sp1, ERE1, and ERE2) drove distinct differential expression in each cell type. Sp1 protein was expressed in a menstrual-cycle stage-specific expression pattern in endometrium, first expressed in perivascular cells., Conclusions: Tissue specificity inherent to a regulatory element as well as differential cellular expression of transcription factors imparts differential tissue-specific estrogen responsiveness.

Breast cancer is the most common non-skin cancer among women in this country. Breast cancer risk is significantly influenced by genetics, but over 70% of the women that are diagnosed have noninherited or sporadic cancer. The risk of breast cancer is thought to be modified by lifestyle and environment. Exposures to certain chemicals and hormone-mimicking or endocrine-disrupting compounds (EDCs) are suspected of contributing to increased breast cancer incidence as well as precocious puberty in the United States. Studies of EDC effects in rodents indicate that multiple toxicants can alter mammary gland development, with or without changing other markers of puberty. EDCs can cause transient and persistent effects on mammary gland development depending on dose, exposure parameters, and whether exposure was during critical periods of gland growth or differentiation. Adverse effects from these abnormal developmental patterns include the presence of carcinogen-sensitive structures in greater numbers or for longer periods in the gland and inhibited functional differentiation leading to malnutrition or increased mortality of their offspring. Developmental toxicants of the mammary gland could lead to an increase in the incidence of mammary tumors if they alter circulating or tissue-localized hormone levels, gland receptor expression patterns, hormone transport, or metabolism that results in altered response to endogenous hormones or growth factors. Environmental disruptors of rodent mammary gland development must be identified for informed decisions in epidemiological studies aimed at identification of environmental factors contributing to breast cancer risk, altered breast development during puberty, or inability to produce sufficient breast milk.

Background: Increased mammographic density reduces the sensitivity of screening mammography, is associated with increased breast cancer risk, and may be hormone related. We assessed the effect of estrogen-plus-progestin therapy on mammographic density., Methods: In a racially and ethnically diverse ancillary study of the Women's Health Initiative, we examined data from 413 postmenopausal women who had been randomly assigned to receive daily combined conjugated equine estrogens (0.625 mg) plus medroxyprogesterone acetate (i.e., progestin; 2.5 mg) (n = 202) or daily placebo (n = 211). We assessed the effect of estrogen plus progestin on measured mammographic percent density and abnormal findings over a 1-year and 2-year period. All tests of statistical significance were two-sided and were based on F tests or t tests from mixed-effects models., Results: Mean mammographic percent density increased by 6.0% at year 1, compared with baseline, in the estrogen-plus-progestin group but decreased by 0.9% in the placebo group (difference = 6.9%, 95% confidence interval [CI] = 5.3% to 8.5%; P < .001). The mean changes in mammographic density persisted but were attenuated slightly after 2 years, with an absolute increase of 4.9% in the estrogen-plus-progestin group and a decrease of 0.8% in the placebo group (difference = 5.7%, 95% CI = 4.3% to 7.3%; P < .001). These effects were consistent across racial/ethnic groups but were higher among women aged 70-79 years in the estrogen-plus-progestin group (mean increase at year 1 = 11.6%) than in the placebo group (mean decrease at year 1 = 0.1%) (difference of the means = 11.7%, 95% CI = 8.2% to 15.4%; P < .001, comparing across age groups). At year 1, women who were adherent to treatment in the estrogen-plus-progestin group had a mean increase in density of 7.7% (95% CI = 5.9% to 9.5%), and women in the placebo group had a mean decrease in density of 1.1% (95% CI = 0.3% to 1.9%). Use of estrogen plus progestin was associated with an increased risk of having an abnormal mammogram at year 1 (relative risk = 3.9, 95% CI = 1.5 to 10.2; P = .003), compared with placebo, that was not explained by an increase in density., Conclusions: Use of up to 2 years of estrogen plus progestin was associated with increases in mammographic density.

Background: The purpose of this study was to examine the effects of duration of hormone therapy (HT) and treatment regimens on mammographic density., Methods: A retrospective study was carried out of of 467 post-menopausal women who received estrogen or estrogen-progestin and had regular mammographic density determination by the Breast Imaging Reporting and Data System between 1994 and 2001., Results: The fraction of women using HT who had an increase in mammographic density became more important over time. Further analysis of the effects of regimens after 4 years of HT shows that the increase in mean density was much greater in women receiving combined HT than in those receiving estrogen alone. The incidence of increased mammographic density showed significantly progressive increases over the duration of combined HT from 7.5 to 22.4%., Conclusions: Although most women using HT maintained breast density at pre-treatment levels, there is a note of caution for women using long-term HT, especially those using combined estrogen-progestin.

Phytochemicals are natural dietary constituents of fruits and vegetables. Some of these phytochemicals are known to affect estrogen-metabolizing enzymes. In breast tissue, estradiol can be metabolized to the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE(2) and 4-OHE(2)). Catechol estrogens are suspected carcinogens potentially involved in the etiology of breast cancer. Catechol-O-methyltransferase (COMT) converts the catechol estrogens to their inactive methoxy derivatives (2-MeOE(2) and 4-MeOE(2)). In this study we investigated the effects of several phytochemicals on COMT activity in cytosolic fractions of seven healthy human mammary tissues from reduction mammoplasty. Large interindividual variations were observed in the constitutive levels of COMT activity. However, in all cytosol samples the catalytic efficiency of COMT was greater for 2-MeOE(2) formation than for 4-MeOE(2) formation. The known COMT inhibitor Ro 41-0960 and several phytochemicals with a catechol structure (quercetin, catechin, and (-)-epicatechin) concentration-dependently inhibited COMT activity, while phytochemicals without a catechol structure (genistein, chrysin, and flavone) showed no effect up to 30 microM. Distinct interindividual variations were observed in sensitivity toward COMT inhibition among the various tissue samples, as was shown by the range in IC(50) values for Ro 41-0960 (5-42 nM). The toxicological relevance of COMT inhibition and the effect of reduced inactivation of catechol estrogens was studied by determining the amount of catechol estrogen-induced DNA damage in MCF-7 cells using the comet assay. Catechol estrogens alone caused no increase of DNA damage compared with control treated cells. However, both Ro 41-0960 and quercetin caused a decrease of methoxy estradiol formation and an increase of catechol estrogen-induced DNA damage in MCF-7 cells. This suggests that phytochemicals with a catechol structure have the potential to reduce COMT activity in mammary tissues and may consequently reduce the inactivation of potentially mutagenic estradiol metabolites and increase the chance of DNA damage.

Androgens have important physiological effects in women. Postmenopausal androgen replacement, most commonly as testosterone therapy, is becoming increasingly widespread. This is despite the lack of clear guidelines regarding the diagnosis of androgen insufficiency, optimal therapeutic doses, and long-term safety data. With respect to the breast specifically, there is the potential for exogenous testosterone to exert either androgenic or indirect estrogenic actions, with the latter potentially increasing breast cancer risk. In experimental studies, androgens exhibit growth-inhibitory and apoptotic effects in some, but not all, breast cancer cell lines. Differing effects between cell lines appear to be due primarily to variations in concentrations of specific coregulatory proteins at the receptor level. In rodent breast cancer models, androgen action is antiproliferative and proapoptotic, and is mediated via the androgen receptor, despite the potential for testosterone and dehydroepiandrosterone to be aromatized to estrogen. The results from studies in rhesus monkeys suggest that testosterone may serve as a natural endogenous protector of the breast and limit mitogenic and cancer-promoting effects of estrogen on mammary epithelium. Epidemiological studies have significant methodological limitations and provide inconclusive results. The strongest data for exogenous testosterone therapy comes from primate studies. Based on such simulations, inclusion of testosterone in postmenopausal estrogen-progestin regimens has the potential to ameliorate the stimulating effects of combined estrogen-progestin on the breast. Research addressing this is warranted; however, the number of women that would be required for an adequately powered randomized controlled trial renders such a study unlikely.

Background: Although mammographic breast density is associated with the risk of breast cancer and is influenced by hormone levels, the effects of tamoxifen on breast density in healthy women and whether tamoxifen-induced density changes are associated with breast cancer risk are unclear. We investigated mammographic breast density in healthy women with an increased risk of breast cancer at baseline and during 54 months of tamoxifen treatment., Methods: Mammograms were reviewed from 818 breast cancer-free women (388 in the tamoxifen group and 430 in the placebo group) at high risk for breast cancer, from the International Breast Cancer Intervention Study I, a trial of tamoxifen for breast cancer prevention. Breast density measurements, at baseline and during treatment, were obtained at 12- to 18-month intervals. Multivariable analysis was used to assess associations with breast density. All statistical tests were two-sided., Results: Breast density at baseline was similar in placebo (42.6%, 95% confidence interval [CI] = 39.6% to 45.6%) and tamoxifen (41.9%, 95% CI = 38.8% to 45.0%) groups. The main determinants of breast density at baseline were age, menopausal status, body mass index, and previous atypical hyperplasia. A greater density reduction in the tamoxifen group (7.9%, 95% CI = 6.9% to 8.9%) than in the placebo group (3.5%, 95% CI = 2.7% to 4.3%) was apparent within 18 months of treatment (P<.001); the reduction in density continued until 54 months of treatment. After 54 months of tamoxifen treatment, breast density was 28.2% (decrease from baseline = 13.7%, 95% CI = 12.3% to 15.1%; P<.001) in the tamoxifen group and 35.3% (decrease from baseline = 7.3%, 95% CI = 6.1% to 8.4%; P<.001) in the placebo group. The tamoxifen-associated density reduction was apparent in all subgroups, but there was a statistically significant interaction with age. In women aged 45 years or younger at entry, the net reduction with tamoxifen was 13.4% (95% CI = 8.6% to 18.1%), whereas in women older than 55 years, it was 1.1% (95% CI = -3.0% to 5.1%)., Conclusion: Tamoxifen treatment was associated with reduction in breast density, most of which occurred during the first 18 months of treatment.

Background: Mammographic density is an independent risk factor for breast cancer. Postmenopausal hormone use is associated with an increase in mammographic density, but the magnitude of the density increase is unknown., Methods: Baseline and 12-month mammograms were obtained for 571 (65%) of the 875 women, aged 45-64 years, who were enrolled in the Postmenopausal Estrogen/Progestin Interventions Trial and randomly assigned to receive placebo, daily conjugated equine estrogens at 0.625 mg/day (CEE), daily CEE and medroxyprogesterone acetate (MPA) at 10 mg/day on days 1-12 (CEE+MPA-cyclic), daily CEE and MPA at 2.5 mg/day (CEE+MPA-continuous), or daily CEE and micronized progesterone (MP) at 200 mg/day on days 1-12 (CEE+MP). We analyzed digitized mammograms to determine the percentage of the left breast that was composed of dense tissue (i.e., mammographic percent density). Linear regression analysis was used to examine the effects of treatments on the change in mammographic percent density between baseline and 12 months, before and after adjustment for possible confounders. All statistical tests were two-sided., Results: The adjusted absolute mean changes in mammographic percent density over 12 months were 4.76% (95% confidence interval [CI] = 3.29% to 6.23%), 4.58% (95% CI = 3.19% to 5.97%), and 3.08% (95% CI = 1.65% to 4.51%) for women in the CEE+MPA-cyclic, CEE+MPA-continuous, and CEE-MP groups, respectively. Each of those absolute mean changes was statistically significantly different from the adjusted absolute mean change in mammographic percent density for women in the placebo group, which was -0.07% (95% CI = -1.50% to 1.38%)., Conclusion: Greater mammographic density was associated with the use of estrogen/progestin combination therapy, regardless of how the progestin was given, but not with the use of estrogen only.

Cumulative exposure to oestrogen has been linked to increased risk of breast cancer. Whilst oestrogens induce cancers in rodent bioassays it is unclear whether the mechanisms involved are genotoxic and/or epigenetic. The cytokinesis block micronucleus (CBMN) and the alkaline single cell-gel electrophoresis 'Comet' assays were used to examine MCF-7 cells for chromosomal damage and DNA single-strand breaks (SSBs), respectively. The comet-forming activities of oestrogens were also tested in a 72 h primary culture of cells isolated from freshly expressed breast milk. Micronuclei (MN) were scored in 500 binucleate cells per treatment and SSBs were quantified by comet tail length (CTL) (microm). Effects on mitotic rate (per cent binucleate cells) and cell viability (per cent plating efficiency) were also assessed. beta-Oestradiol, oestrone and oestriol were tested for genotoxicity in the 10(-10)-10(-4) M and 10(-10)-10(-2) M concentration ranges in the CBMN and Comet assays, respectively. Beta-Oestradiol, following 24 h treatment but not 120 h treatment, induced increases (up to 3-fold) in MN at a concentration of 10(-9) M. Oestrone induced dose-related increases in MN (up to 5-fold) following both 24 and 120 h treatment, whereas oestriol appeared not to induce MN. All three oestrogens induced dose-related increases in per cent binucleate cells suggesting that they enhance mitotic rate. In the Comet assay both beta-oestradiol and oestrone induced dose-related increases in SSBs (up to 7-fold over control CTL) and were significantly comet-forming (P < 0.0001) at concentrations as low as 10(-9) and 10(-8) M, respectively, whereas oestriol was less genotoxic. All three oestrogens were significantly comet-forming (P < 0.0001) in a primary culture of breast milk cells, suggesting that they can damage the target cells from which breast cancers may eventually arise.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.

Soy products containing phytoestrogens have received much attention as dietary components to promote better health. Daidzein, an isoflavone and phytoestrogen component of soy, was investigated for its potential to alter fertility and cause developmental toxicity to the reproductive tract in female rats, for chemoprevention to the mammary gland, and to study its bioavailability. Diets containing 0 mg, 250 mg (low dose), and 1000 mg (high dose) daidzein/kg feed were fed to virgin female rats, starting 2 weeks prior to breeding and continued until the offspring were 50 days postpartum. The serum daidzein concentrations in adult female rats fed the low and high daidzein-containing diets were determined to be 6- and 13-fold higher than serum daidzein concentrations of Asians eating a traditional diet high in soy. Both daidzein doses had no significant effect on fertility, numbers of male and female offspring, and anogenital distances. The high, but not the low, daidzein dose resulted in reduced body weight, a fact that may be explained by reduced feed consumption. Circulating progesterone, but not estrogen, levels were statistically reduced with the high, but not low daidzein-containing diet. Both daidzein doses resulted in slight, but not significant, decreases in ovarian and uterine weights, and mammary gland size. Histomorphological analysis of the reproductive tracts of female offspring 50 days of age exposed perinatally to daidzein did not reveal any pathology in the vaginal, uterine, ovarian, and mammary tissues. Perinatal exposure of female offspring to 250 mg daidzein/kg diet did not alter mammary gland development or ontogeny of chemically induced mammary tumors in rats treated with dimethylbenz(a)anthracene on day 50. With the low dietary daidzein dose, total equol (major metabolite) and daidzein concentrations in the blood of pregnant females, 7-day-old, 21-day-old, and 50-day-old female offspring were 529 and 303 nM, 163 and 982 nM, 1188 and 1359 nM, and 3826 and 630 nM, respectively. With the high daidzein diet, equol and daidzein concentrations in the blood of pregnant females, 7-day-old, 21-day-old, and 50-day-old female offspring were 4462 and 407 nM, 1013 and 3841 nM, 6472 and 3308 nM, and 7228 and 1430 nM, respectively. Eighty-nine to 99% of daidzein and equol were in the conjugated form. In the 21-day-old postconceptus exposed to the low and high daidzein diets, total equol and daidzein blood concentrations were 59 and 34 nM, and 358 and 132 nM, respectively. Virtually all of the daidzein in the milk of 7-day-old rats exposed to the low and high daidzein-containing diet were unconjugated, 2.6 microM and 7.3 microM, respectively. Total milk equol concentrations were 654 nM and 3.8 microM, of which 94% and 44% were unconjugated. In mammary glands of 7-day-old offspring exposed to 250 mg daidzein/kg diet, total daidzein concentrations were 407 nM (98% aglycone). We conclude that supraphysiological concentrations of daidzein administered via the diet did not cause significant toxicity to the female reproductive tract or provide a protective effect against chemically induced mammary cancer.

The recognition of selective estrogen receptor modulation in the laboratory has resulted in the development of two selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, for clinical application in healthy women. SERMs are antiestrogenic in the breast but estrogen-like in the bones and reduce circulating cholesterol levels. SERMs also have different degrees of estrogenicity in the uterus. Tamoxifen is used specifically to reduce the incidence of breast cancer in premenopausal and postmenopausal women at risk for the disease. In contrast, raloxifene is used specifically to reduce the risk of osteoporosis in postmenopausal women at high risk for osteoporosis. The study of tamoxifen and raloxifene (STAR) trial is currently comparing the ability of these SERMs to reduce breast cancer incidence in high-risk postmenopausal women. There is intense interest in understanding the molecular mechanism(s) of action of SERMs at target sites in a woman's body. An understanding of the targeted actions of this novel drug group will potentially result in the introduction of new multifunctional medicines with applications as preventive agents or treatments of breast cancer and endometrial cancer, coronary heart disease, and osteoporosis.

These experiments tested whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters mammary gland differentiation, estrogen receptor alpha (ERalpha) expression levels, or the response to estrogen in the female postpubertal rat mammary gland. Pregnant Holtzman rats were administered a single oral dose of 1 microg/kg TCDD or vehicle on gestation-day 15. Exposed and non-exposed female offspring were weaned on postnatal day 21 and ovariectomized at 9 weeks of age. Two weeks later, both TCDD and control animals were divided into 3 groups, receiving treatment with placebo, 0.025, or 0.1 mg 17beta-estradiol pellet implants. After 48 h, mammary tissue was removed for analysis following euthanasia. TCDD-exposed mammary glands demonstrated impaired differentiation as measured by the distribution of terminal ductal structures and increased expression levels of ERalpha. The response to exogenous estrogen was tested in TCDD-exposed animals and compared to control non-exposed animals. Estrogen stimulation of the TCDD-exposed glands induced progesterone receptor expression and mammary gland differentiation as measured by a shift in distribution from terminal end buds and terminal ducts to Types I and II lobules. Control glands were better differentiated at baseline and did not exhibit any significant changes in the distribution of terminal ductal structures following estrogen stimulation. The increase in progesterone receptor-expression levels by exogenous estrogen in control glands was similar to the TCDD-exposed glands. These experiments demonstrate that in utero and lactational exposures to TCDD impair mammary gland differentiation but that TCDD-exposed mammary glands retain the ability to differentiate in response to estrogen.

Overexpression of des(1-3) human insulin-like growth factor I (IGF-I) in the mammary glands of transgenic mice (WAP-DES) inhibits apoptosis during natural, but not forced, mammary involution. We hypothesized that this differential response would correlate with the expression of IGF signal transducers. Forced and natural involution were analyzed in nontransgenic and WAP-DES mice beginning on day 16 postpartum. During natural involution, mammary gland wet weight was higher and apoptosis was lower in WAP-DES than in nontransgenic mice. The WAP-DES transgene had no effect on these parameters during forced involution. Mammary tissue concentrations of the transgene protein were 2- to 10-fold higher than those of endogenous IGF-I. Western blot analysis of pooled mammary tissue extracts demonstrated only slightly higher phosphorylation of the IGF signal transducers insulin receptor substrate-1 (IRS-1) and Akt in the WAP-DES than in nontransgenic mice. Dramatic early reductions in phospho-IRS-1, phospho-Akt, IRS-1, IRS-2, and Akt proteins occurred during forced, but not natural, involution. The abundance of the IGF-I receptor and the messenger RNAs for the IGF-I receptors, IRS-1 and -2, were not affected by either genotype or involution. These findings support the conclusions that mammary cells lose their responsiveness to insulin-like signals during forced involution, and that posttranscriptional or posttranslational regulation of IRS-1 and IRS-2 may play a role in this loss.

Background: High mammographic density is associated with increased breast cancer risk. Previous studies have shown that estrogens increase breast density on mammograms, but the effect on mammographic density of selective estrogen receptor modulators, such as raloxifene, is unknown. We assessed changes in mammographic density among women receiving placebo, raloxifene, or conjugated equine estrogens in an osteoporosis prevention trial., Methods: In a 5-year multicenter, double-blind, randomized, placebo-controlled osteoporosis prevention trial, healthy postmenopausal women who had undergone hysterectomy less than 15 years before the study and had no history of breast cancer received placebo, raloxifene (at one of two doses), or conjugated estrogens (ERT). Women from English-speaking investigative sites who had baseline and 2-year craniocaudal mammograms with comparable positioning (n = 168) were eligible for this analysis. Changes in mammographic density were determined by digital scanning and computer-assisted segmentation of mammograms and were analyzed with the use of analysis of variance. All statistical tests were two-sided., Results: Among the four treatment groups after 2 years on study, the mean breast density (craniocaudal view) was statistically significantly greater in the ERT group than it was in the other three groups (P<0.01 for all three comparisons). Within treatment groups, the mean breast density from baseline to 2 years decreased statistically significantly in women receiving the placebo or either the higher or lower raloxifene dose (P = 0.003, P = 0.002, and P<0.001, respectively) and showed a nonstatistically significant increase in women receiving ERT., Conclusions: In an osteoporosis prevention trial, raloxifene did not increase breast density after 2 years of treatment. Raloxifene administration should not interfere with, and could even enhance, mammographic detection of new breast cancers.

Background: A major limiting factor in human cancer chemotherapy is toxicity in normal tissues. Our goal was to determine whether normal proliferating cells could be protected from chemotherapeutic agents by taking advantage of the differential drug sensitivity of cell cycle G(1) checkpoint in normal and cancer cells., Methods: Normal mammary epithelial cells and mammary cancer cells were initially treated with staurosporine at a cytostatic (i.e., nonlethal) concentration, which preferentially arrests normal cells in the G(0)/G(1) phase of the cell cycle without affecting the proliferation of tumor cells. After the selective arrest of normal cells in G(0)/G(1), both normal and tumor cells were treated with doxorubicin or camptothecin, two cytotoxic (i.e., lethal) chemotherapeutic agents. Cells were then allowed to recover in drug-free medium for 12 days., Results: After pretreatment of both normal and tumor cells with staurosporine followed by treatment with doxorubicin or camptothecin, tumor cells were selectively killed by chemotherapeutic agents, whereas normal cells resumed proliferation after the drugs were removed. Pretreatment with staurosporine also protected normal circulating lymphocytes that had been induced to proliferate in vitro with phytohemagglutinin from chemotherapeutic agents. Staurosporine-induced arrest of normal cells in G(0)/G(1) phase was reversible, and arrested cells tolerated doses of camptothecin that were more than 100-fold higher than necessary to eradicate all tumor cells in culture. Staurosporine-mediated G(0)/G(1) arrest targets the retinoblastoma protein (pRb) pathway and was accompanied by a rapid decrease in cyclin-dependent kinase (CDK) 4 protein levels, increased binding of CDK inhibitors p21 and p27 to CDK2, and inhibition of CDK2 activity in normal cells., Conclusions: Breast cancer cells with defective checkpoints regulated by the pRb pathway can be targeted specifically with chemotherapeutic agents, following staurosporine-mediated, selective and reversible G(0)/G(1) arrest in normal cells.

To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HC11 cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.

Background: Mammographic parenchymal patterns are of particular interest because the denser patterns reduce screening sensitivity as well as increasing breast cancer risk, and because they have been shown to be affected by exogenous oestrogens., Methods: We designed a case-control study comprising 200 cases with high-risk (P2 and DY) pattern and 200 controls with low-risk (N1 and P1) pattern. Mammograms were evaluated according to the Wolfe classification., Results: Parity, body mass index (BMI) and current smoking were inversely and independently associated, whereas late age of menarche and history of benign breast disease were positively associated with high-risk mammographic patterns. Current-users of hormone replacement therapy (HRT) were more than twice as likely to have a high-risk pattern than never-users (OR = 2.48, 95% CI : 1.32-4.61). Women who used HRT for more than 5 years were almost three times more likely to have a high-risk pattern than never-users (OR = 2.77, 95% CI : 1.11-6.91). Relative to never-users, women who started HRT before the menopause were more than twice as likely to have a high-risk pattern (OR = 2.53, 95% CI : 1.31-4.87)., Conclusions: Careful clinical and mammographical follow-up might be appropriate in women undergoing HRT. The HRT-induced mammographic pattern might be regarded as a new baseline and changes with respect to this new pattern could then be monitored over time.

Background: Overexpression of the gene c-erbB2, which encodes a receptor tyrosine kinase, in breast tumors has been linked with either increased or decreased response of breast cancer patients to various therapies. In breast cancer cell lines, overexpression of exogenous c-erbB2 sometimes alters drug sensitivities but sometimes has no effect. To avoid the genetic complexities associated with established cancer cell lines, normal human mammary epithelial cells (HMECs) were studied to determine whether c-erbB2 overexpression by itself would alter chemosensitivity., Methods: HMECs were designed to overexpress c-erbB2, and these cells were then evaluated for alterations in chemosensitivity., Results: HMECs overexpressing c-erbB2 failed to show any alterations in chemosensitivity to a panel of chemotherapeutic agents, as indicated by 95% confidence intervals on growth curves of cells treated with or without the agent of interest. With the use of fluorescence-activated cell sorting to enrich for HMECs overexpressing c-erbB2 on their surface, an 85% pure population of cells was isolated and their chemosensitivity was evaluated. Again, the cells failed to display any alterations in chemosensitivity., Conclusions: These results suggest that overexpression of c-erbB2 is not sufficient by itself to induce changes in chemosensitivity. Cellular studies using normal human cells in which the complexity of the system can be carefully controlled by the addition of one, two, or even more genes associated with cancer development may provide valuable information about how the products of the genes interact with each other and which combinations are critical in regulating chemosensitivity.

Most human mammary carcinomas originate in the epithelial cells of the breast ducts. A potential role of heterocyclic amines (HAs) in the aetiology of this disease has led us to investigate peroxidase-catalysed and stromal (non-epithelial) activation of the HA 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which may subsequently lead to DNA damage in the adjacent human mammary epithelial cells (HMECs). HAs are formed when proteinaceous foods are cooked at high temperature and some, but not all, can cause mammary tumours in rats. Myeloperoxidase (MPO) and lactoperoxidase (LPO) are peroxidase enzymes present in breast secretions. (32)P-post-labelling analysis showed that IQ-DNA adducts were formed after co-incubation of IQ (500 microM) with calf thymus DNA, hydrogen peroxide and either bovine LPO or horseradish peroxidase (HRP). The major HRP-mediated IQ-DNA adduct co-migrated on TLC and HPLC with the major adduct formed in HMECs, suggesting a common reactive intermediate (nitrenium ion). IQ-DNA adducts were also formed in extracellular DNA when phorbol myristate acetate-stimulated neutrophils (which activate IQ via MPO) were co-incubated with IQ (500 microM) and extracellular plasmid (4 +/- 1 adducts/10(8) nucleotides) or calf thymus DNA (6 +/- 2). Mean adduct formation was five to seven times greater in neutrophil DNA (31 +/- 20). Primary cultures of human mammary fibroblasts or epithelial cells isolated from reduction mammoplasty tissues (n = 4 individuals) were incubated with IQ (500 microM) and formed 2.5 and 14.8 adducts/10(8) nucleotides (mean values), respectively. Our results indicate the possible contribution of stromal cells and breast peroxidases to the metabolic activation of carcinogens in the mammary gland.