Endocrine Disruptors and Hypothalamic Sexual Differentiation

The so-called endocrine disruptors have been described as compounds which interfere with the estrogen action in their receptors and may exert a crucial role in the development of the reproductive tract and in the brain sexual differentiation. Thus, conducts and/or exposure to these drugs in the perinatal period that apparently do not endanger the neonate may cause side effects. During embrionary development, the gonads, through discharge of a small quantity of reproductive hormones, will guarantee the phenotype of male or female at birth, as well as actuate in specific areas sexual differentiation of the central nervous system. Several experimental models have shown an interference of drugs acting as endocrine disruptors in hypothalamic sexual differentiation. Thus, reproductive function is impaired by exposure to estrogen in the perinatal life of rats and the mechanisms involved in this effect are distinct for males and females. Perinatal exposure to drugs which may be considered endocrine disrupters may induce an incomplete masculinization and defeminization of the central nervous system. Alterations in these processes, if present, generally are perceived only at puberty or adult reproductive life. These later alterations may include anomalies in the process of fertility or in sexual behavior.

Efeito do raloxifeno sobre a densidade mamográfica em mulheres na pós-menopausa

PURPOSE: to evaluate changes in mammographic breast density in postmenopausal women using raloxifene. METHODS: in this clinical trial, 80 women (mean age=61.1 years) were studied prospectively. Forty patients received 60 mg/day raloxifene, and 40 women comprised the non-treated group (control), paired by age and time of menopause. The treated group was composed of patients with osteoporosis of the lumbar spine. Those with history of breast surgery and users of hormone therapy up to six months prior to the study were excluded. The breast density was assessed qualitatively (subjective) and quantitatively (objective) in two moments, initial and final, after a 6-month follow-up. The 320 mammograms (craniocaudal and oblique) were interpreted qualitatively by the Breast Imaging Reporting and Data System (BI-RADS) classification and quantitatively by digital scanning and computer-assisted segmentation. For statistical analysis t-test, Wilcoxon Mann-Whitney, Spearman correlation and the kappa index were used. RESULTS: on the initial statistical comparison, the groups were considered homogenous for the variables: analyzed age, time of menopause, parity, breast feeding, previous hormonal therapy and body mass index. Baseline breast density, by qualitative and quantitative methods, correlated negatively with the age in both groups (p<0.05). Concerning the other variables, there was no correlation. After six months, no alteration was observed in the mammographic breast density in 38 women of raloxifene group and 38 of the control group, by qualitative method. However, by quantitative method, no alteration was observed in 30 women of the raloxifene group and 27 controls (p>0.05). It was observed a weak agreement rate (kappa=0.25) between the BI-RADS classification and digital scanning/computer-assisted segmentation. CONCLUSIONS: in post-menopausal women with osteoporosis, submitted to raloxifene treatment for six months, no alterations were observed on the mammographic breast density.

Chronic alcohol intake upregulates hepatic expression of carotenoid cleavage enzymes and PPAR in rats

Excessive and chronic alcohol intake leads to a lower hepatic vitamin A status by interfering with vitamin A metabolism. Dietary provitamin A carotenoids can be converted into vitamin A mainly by carotenoid 15,15′-monooxygenase 1 (CMO1) and, to a lesser degree, carotenoid 9′10′-monooxygenase 2 (CMO2). CMO1 has been shown to be regulated by several transcription factors, such as the PPAR, retinoid X receptor, and thyroid receptor (TR). The regulation of CMO2 has yet to be identified. The impact of chronic alcohol intake on hepatic expressions of CMO1 and CMO2 and their related transcription factors are unknown. In this study, Fischer 344 rats were pair-fed either a liquid ethanol Lieber-DeCarli diet (n = 10) or a control diet (n = 10) for 11 wk. Hepatic retinoid concentration and expressions of CMO1, CMO2, PPARγ, PPARα, and TRβ as well as plasma thyroid hormones levels were analyzed. We observed that administering alcohol decreased hepatic retinoid levels but increased mRNA concentrations of CMO1, CMO2, PPARγ, PPARα, and TRβ and upregulated protein levels of CMO2, PPARγ, and PPARα. There was a positive correlation of PPARγ with CMO1(r = 0.89; P<0.0001) and both PPARγ and PPARα with CMO2 (r = 0.72, P< 0.001 and r = 0.62, P< 0.01, respectively). Plasma thyroid hormone concentrations did not differ between the control rats and alcohol-fed rats. This study suggests that chronic alcohol intake significantly upregulates hepatic expression of CMO1 and, to a much lesser extent, CMO2. This process may be due to alcohol-induced PPARγ expression and lower vitamin A status in the liver. © 2010 American Society for Nutrition.

Fine needle aspirate cell blocks are reliable for detection of hormone receptors and HER-2 by immunohistochemistry in breast carcinoma

Aims: To evaluate the reliability of fine needle aspirate cell blocks in the assessment of oestrogen receptor (ER), progesterone receptor (PR) and HER-2/neu proteins by immunohistochemistry in comparison with surgical specimens. Materials and methods: This is a retrospective study of 62 cases of breast carcinoma diagnosed by fine needle aspiration cytology (FNAC) and confirmed using the surgical specimen. Immunohistochemical tests were performed to assess the presence of oestrogen receptor (ER), progesterone receptor (PR) and HER-2/neu proteins in cell blocks and the corresponding surgical specimens. The cell block method used alcohol prior to formalin fixation. Cases with 10% or more stained cells were considered positive for ER and PR. Positivity for HER-2/neu was assessed on a scale of 0-3+. The criterion for positivity was a score of 3+. Results: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the cell blocks in the investigation of ER, PR and HER-2/neu protein (3+) were (%): ER, 92.7, 85.7, 92.7, 85.7 and 90.3; PR, 92.7, 94.7, 97.4, 87.0 and 93.5; HER-2/neu, 70.0, 100.0, 100.0, 94.5 and 95.2. Discrepancies were seen in cell blocks in the 1+ and 2+ HER-2/neu staining scores: two of 12 cases scoring 2+ and one case of 26 scoring 1+ on cell blocks scored 3+ on surgical specimens. The correlation index between cell block and corresponding surgical specimen varied from 90% to 94%. Conclusion: Cell blocks provide a useful method of assessing ER, PR and HER-2/neu, mainly for inoperable and recurrent cases, but consideration should be given to carrying out FISH analysis on 1+ as well as 2+ HER-2/neu results. © 2012 Blackwell Publishing Ltd.

Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. © 2013 Moura et al; licensee BioMed Central Ltd.

Risk of metabolic syndrome in postmenopausal breast cancer survivors

The aim of this study was to assess the risk of metabolic syndrome (MetS) in postmenopausal breast cancer survivors as compared with postmenopausal women without breast cancer. METHODS: In this cross-sectional study, 104 postmenopausal breast cancer survivors were compared with 208 postmenopausal women (controls) attending a university hospital. Eligibility criteria included the following: amenorrhea longer than 12 months and aged 45 years or older, treated for breast cancer, and metastasis-free for at least 5 years. The control group consisted of women with amenorrhea longer than 12 months and aged 45 years or older and without breast cancer, matched by age and menopause status (in a proportion of 1:2 as sample calculation). Clinical and anthropometric data were collected. Biochemical parameters, including total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, and C-reactive protein, were measured. Women showing three or more diagnostic criteria were diagnosed as having MetS: waist circumference of 88 cm or larger, blood pressure of 130/85 mm Hg or higher, triglycerides level of 150 mg/dL or higher, high-density lipoprotein cholesterol level lower than 50 mg/dL, and glucose level of 100 mg/dL or higher. For statistical analysis, Student's t test, χ2 test, and logistic regression (odds ratio [OR]) were used. RESULTS: The mean (SD) age of breast cancer survivors was 60.6 (8.6) years, with a mean (SD) follow-up of 9.4 (4.4) years. A higher percentage of breast cancer survivors (46.2%) were obese as compared with controls (32.7%; P < 0.05), and a smaller percentage showed optimal values for low-density lipoprotein cholesterol, glucose, and C-reactive protein versus controls (P < 0.05). MetS was diagnosed in 50% of breast cancer survivors and in 37.5% of control group women (P < 0.05). Among the MetS diagnostic criteria, the most prevalent was abdominal obesity (waist circumference >88 cm), affecting 62.5% and 67.8% of the participants, respectively. In the control group, breast cancer survivors had a higher risk for MetS (OR, 1.66; 95% CI, 1.04-2.68), dysglycemia (OR, 1.05; 95% CI, 1.09-3.03), and hypertension (OR, 1.71; 95% CI, 1.02-2.89). CONCLUSIONS: Postmenopausal breast cancer survivors present a higher risk of developing MetS as compared with women without breast cancer. © 2012 by The North American Menopause Society.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen

Background: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM.Methods: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm3) and the proportions of each tumor that were alive, necrotic or degenerative (mm2). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers.Results: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment.Conclusions: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors. © 2013 Dias et al.; licensee BioMed Central Ltd.

Role of brain nitric oxide in the cardiovascular control of bullfrogs

The goal of the present study was to determine if nitric oxide (NO) acting on the brain of bullfrog (Lithobates catesbeianus) is involved in arterial pressure and heart rate (HR) control by influencing sympathetic activity. We investigated the effect of intracerebroventricular injections of l-NMMA (a nonselective NO synthase inhibitor) on mean arterial blood pressure (MAP), HR and cutaneous vascular conductance (CVC) of pelvic skin after intravenous injection of α or β adrenergic blockers, prazosin or sotalol, respectively. Arterial pressure was directly measured by a telemetry sensor inserted in the aortic arch of animals. l-NMMA increased MAP, but did not change HR. This hypertensive response was inhibited by the pre-treatment with prazosin, but accentuated by sotalol. The effect of l-NMMA on MAP was also inhibited by i.v. injections of the ganglionic blocker, hexamethonium. Thus, NO acting on the brain of bullfrog seems to present a hypotensive effect influencing the sympathetic activity dependent on α and β adrenergic receptors in the periphery. © 2013 Elsevier Inc.

Clinicopathological significance of ERCC1 expression in breast cancer

The excision repair cross-complementation 1 (ERCC1) enzyme plays an essential role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in different types of cancer. The aim of the present study was to evaluate the clinicopathological significance of ERCC1 expression in breast cancer patients. We analyzed the immunohistochemical expression of ERCC1 in a tissue microarray from 135 primary breast carcinomas and correlated the immunohistochemical findings with clinicopathological factors and outcome data. ERCC1 expression analysis was available for 109 cases. In this group, 58 (53.2%) were positive for ERCC1. ERCC1-positive expression was correlated with smaller tumor size (P=0.007) and with positivity for estrogen receptor (P=0.040), but no correlation was found with other clinicopathological features. Although not statistically significant, triple negative breast cancers were more frequently negative for ERCC1 (61.5% of the cases) compared to the non-triple negative breast cancer cases (41.5%). In conclusion, ERCC1 expression correlated significantly with favorable prognostic factors, such as smaller tumor size and ER-positivity, suggesting a possible role for ERCC1 as a predictive and/or prognostic marker in breast cancer. © 2013 Elsevier GmbH.

Noradrenergic neurotransmission within the bed nucleus of the stria terminalis modulates the retention of immobility in the rat forced swimming test

The bed nucleus of the stria terminalis (BNST) is a limbic structure that has a direct influence on the autonomic, neuroendocrine, and behavioral responses to stress. It was recently reported that reversible inactivation of synaptic transmission within this structure causes antidepressant-like effects, indicating that activation of the BNST during stressful situations would facilitate the development of behavioral changes related to the neurobiology of depression. Moreover, noradrenergic neurotransmission is abundant in the BNST and has an important role in the regulation of emotional processes related to the stress response. Thus, this study aimed to test the hypothesis that activation of adrenoceptors within the BNST facilitates the development of behavioral consequences of stress. To investigate this hypothesis, male Wistar rats were stressed (forced swimming, 15 min) and 24 h later received intra-BNST injections of vehicle, WB4101, RX821002, CGP20712, or ICI118,551, which are selective α1, α2, β1, and β2 adrenoceptor antagonists, respectively, 10 min before a 5-min forced swimming test. It was observed that administration of WB4101 (10 and 15 nmol), CGP20712 (5 and 10 nmol), or ICI118,551 (5 nmol) into the BNST reduced the immobility time of rats subjected to forced swimming test, indicating an antidepressant-like effect. These findings suggest that activation of α1, β1, and β2 adrenoceptors in the BNST could be involved in the development of the behavioral consequences of stress. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Immunohistochemical investigation of the angiogenic proteins VEGF, HIF-1α and CD34 in invasive ductal carcinoma of the breast

The expression of prognostic markers in cancer has become important in diagnostic routine and research. A high mitotic rate compromises the individual cell access to oxygen and nutrients, due to reduced blood supply. Cells undertake adaptive measures such as vascular endothelial growth factor (VEGF), expressed under the control of hypoxia-inducible factor-1α (HIF-1α). CD34 is an endothelial marker which can show the presence and distribution of blood vessels. This study evaluated the presence and relative expression of VEGF, HIF-1α and CD34 using immunohistochemistry of 60 breast tumors and double staining, correlating the findings with clinical and pathological variables. High VEGF expression was correlated with low cell proliferation, lymph node-negative, smaller tumor size and patients not receiving hormone therapy. High HIF-1α expression predominated in younger (<50-year) patients, subjected to neo-adjuvant therapy and in p53-negative tumors. Absence of metastasis, radiotherapy or hormone treatment, and estrogen receptor (ER)-positive tumors showed high CD34 immunoreactivity. We suggest that the angiogenic factors VEGF, HIF-1α and CD34 are important in breast cancer progression and their abundance in breast tumors has prognostic and predictive value. Crown Copyright © 2013.

Evaluation of estrogenic, antiestrogenic and genotoxic activity of nemorosone, the major compound found in brown Cuban propolis

Background: Brown propolis is the major type of propolis found in Cuba; its principal component is nemorosone, the major constituent of Clusia rosea floral resins. Nemorosone has received increasing attention due to its strong in vitro anti-cancer action. The citotoxicity of nemorosone in several human cancer cell lines has been reported and correlated to the direct action it has on the estrogen receptor (ER). Breast cancer can be treated with agents that target estrogen-mediated signaling, such as antiestrogens. Phytoestrogen can mimic or modulate the actions of endogenous estrogens and the treatment of breast cancer with phytoestrogens may be a valid strategy, since they have shown anti-cancer activity.Methods: The aim of the present investigation was to assess the capacity of nemorosone to interact with ERs, by Recombinant Yeast Assay (RYA) and E-screen assays, and to determine by comet assay, if the compound causes DNA-damaging in tumoral and non-tumoral breast cells.Results: Nemorosone did not present estrogenic activity, however, it inhibited the 17-β-estradiol (E2) action when either of both methods was used, showing their antiestrogenicity. The DNA damage induced by the benzophenone in cancer and normal breast cells presented negative results.Conclusion: These findings suggest that nemorosone may have therapeutic application in the treatment of breast cancer. © 2013 Camargo et al.; licensee BioMed Central Ltd.

Cross-talk with β2-adrenoceptors enhances ligand affinity properties from endothelial alpha1D-adrenoceptors that mediates carotid relaxation

Objectives Our main objectives were to investigate the affinity properties of endothelial and muscular α1D-adrenoceptors and to characterize the cross-talk between endothelial α1D- adrenoceptors and β2-adrenoceptors in rat carotid. Methods Relaxation and contraction concentration-response curves for phenylephrine (α1-adrenergic agonist) were obtained in carotid rings in absence or presence of increasing concentrations of BMY7378 (α 1D-adrenergic antagonist), combined or not with increasing concentration of ICI-118,551 (β2-adrenergic antagonist). Schild analysis was used to estimate the affinity constant from pA2 values of BMY7378. Key Findings BMY7378 produced an unsurmountable antagonism on phenylephrine-induced relaxation but a surmountable antagonism on phenylephrine-induced contraction. BMY7378 potency was higher in inhibiting the relaxation than the contraction induced by phenylephrine because the rightward shifts induced by BMY7378 were greater in the relaxation. The apparent pA 2 value for BMY7378 in phenylephrine-induced relaxation was greater than in contraction. When combined with ICI-118,551, BMY7378 yielded a surmountable antagonism on phenylephrine-induced relaxation and presented a pA2 value similar to that obtained in phenylephrine-induced contraction. Conclusions Endothelial α1D-adrenoceptors, which mediates rat carotid relaxation, present high ligand affinity because of the cross-talk with β2-adrenoceptors, which explains the higher potency of phenylephrine in inducing relaxation than contraction and the atypical unsurmountable antagonism produced by BMY7378 on phenylephrine-induced relaxation. © 2013 Royal Pharmaceutical Society.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

Evaluation of Estrogenic Potential of Flavonoids Using a Recombinant Yeast Strain and MCF7/BUS Cell Proliferation Assay

Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA) and the MCF-7 proliferation assay (E-screen), since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid. © 2013 Resende et al.