Plasma Estrogen Research Articles

Derivation of a Bisphenol a Oral Reference Dose (RfD) and Drinking-Water Equivalent Concentration*

Human exposure to bisphenol A (BPA) is due to that found in the diet, and BPA and its metabolites were detected at parts per billion (or less) concentrations in human urine, milk, saliva, serum, plasma, ovarian follicular fluid, and amniotic fluid. Adverse health effects in mice and rats may be induced after parenteral injection or after massive oral doses. Controlled ingestion trials in healthy adult volunteers with 5 mg d 16-BPA were unable to detect parent BPA in plasma despite exquisitely sensitive (limit of detection = 6 nM) methods, but by 96 h 100% of the administered dose was recovered in urine as the glucuronide. The extensive BPA glucuronidation following ingestion is not seen after parenteral injection; only the parent BPA binds plasma proteins and estrogen receptors (ER). The hypothesis that BPA dose-response may be described by a J- or U-shape curve was not supported by toxicogenomic data collected in fetal rat testes and epididymes (after repeated parenteral exposure at 2–400,000 μg/kg-d), where a clear monotonic dose-response both in the numbers of genes and magnitude of individual gene expression was evident. There is no clear indication from available data that the BPA doses normally consumed by humans pose an increased risk for immunologic or neurologic disease. There is no evidence that BPA poses a genotoxic or carcinogenic risk and clinical evaluations of 205 men and women with high-performance liquid chromatography (HPLC)-verified serum or urinary BPA conjugates showed (1) no objective signs, (2) no changes in reproductive hormones or clinical chemistry parameters, and (3) no alterations in the number of children or sons:daughters ratio. Results of benchmark dose (BMD10 and BMDL10) calculations and no-observed-adverse-effect level (NOAEL) inspections of all available and reproducible rodent studies with oral BPA found BMD and NOAEL values all greater than the 5 mg/kg-d NOAELs from mouse and rat multigeneration reproduction toxicity studies. While allometric and physiologically based pharmacokinetic (PBPK) models were constructed for interspecies scaling of BPA and its interaction with ER, multigeneration feeding studies with BPA at doses spanning 5 orders of magnitude failed to identify signs of developmental toxicity or adverse changes in reproductive tract tissues; the 5-mg/kg-d NOAELs identified for systemic toxicity in rats and mice were less than the oral NOAELs for reproductive toxicity. Thus, it is the generalized systemic toxicity of ingested BPA rather than reproductive, immunologic, neurobehavioral, or genotoxic hazard that represents the point of departure. Using U.S. Environmental Protction Agency (EPA) uncertainty factor guidance and application of a threefold database uncertainty factor (to account for the fact that the carcinogenic potential of transplacental BPA exposure has yet to be fully defined and comprehensive neurobehavioral and immunotoxicologic evaluations of BPA by relevant routes and at relevant doses have yet to be completed) to the administered dose NOAEL results in an oral RfD of 0.016 mg/kg-d. Assuming the 70-kg adult consumes 2 L of water each day and adopting the default 20% U.S. EPA drinking water relative source contribution yields a 100 μg/L BPA total allowable concentration (TAC).

Cyclic changes in endometrial echotexture of cows using a computer-assisted program for the analysis of first- and second-order grey level statistics of B-Mode ultrasound images

The aim of this study was to test the suitability of a computer-assisted echotexture analysis program for analysing first- and second-order grey level statistics of grey levels of B-Mode ultrasound images to examine morphologic changes in the endometrium during oestrous cycle in cows. Four Simmental cows were examined for two consecutive oestrous cycles. Echotexture of the endometrium was assessed by mean grey level (MGL) and homogeneity (HOM) of digitised B-Mode images of the uterine body and both uterine horns. As there were no differences ( P > 0.05) in MGL and HOM, respectively, between the images of the uterine body and the uterine horns, the mean values of all endometrial images were used for subsequent analyses of MGL and HOM. The factor ‘day of oestrous cycle’ showed a highly significant ( P < 0.0001) effect and the factor ‘cow’ showed a significant ( P < 0.05) effect on MGL and HOM. No differences ( P > 0.05) in both echotexture parameters were measured between oestrous cycles within cows. MGL was negatively related to HOM ( r = −0.66; P < 0.0001). Low MGL and high HOM occurred on Day 0 (= ovulation) and between Days −3 and −1. While MGL was consistently ( P > 0.05) low between Days −3 and −1, significant changes of HOM with maximum levels on Day −2 ( P < 0.05) were observed during this time. MGL was consistently ( P > 0.05) high between Days 4 and 13 while HOM was consistently ( P > 0.05) low between Days 2 and 13. From Day −3 to Day −1 ( r = 0.48; P < 0.05) plasma oestrogen levels were correlated with HOM, but not with MGL ( P < 0.05). The results of this study show that changes in endometrial morphology of cows can be measured using a computer-assisted texture analysis program.

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E 1), estradiol (E 2) and estrone sulfate (E 1S) levels in the ultra-low range. Following incubation with [ 3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E 1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E 1, E 2) were separated by chromatography (LH-20). Estrone and E 1S (following hydrolysis) were converted into E 2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[ 125I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E 1, E 2 and E 1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E 1, 0.67 pmol/L for E 2, and 0.55 pmol/L for E 1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E 1, 5.1% for E 2 and 6.1% for E 1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E 1, E 2, and E 1S, respectively. Considering normal plasma levels for E 2 (15 pmol/L), E 1 (80 pmol/L) and E 1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E 2, E 1 and E 1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.

Memory impairments with adjuvant anastrozole versus tamoxifen in women with early-stage breast cancer

Hormones have been implicated as modulators of cognitive functioning. For instance, results of our previous work in women with breast cancer showed that cognitive impairment was more severe and involved more memory domains in those who received adjuvant tamoxifen therapy compared with women who received chemotherapy alone or no adjuvant therapy. Recently aromatase inhibitors such as anastrozole have been used in lieu of tamoxifen for the adjuvant treatment of postmenopausal women with hormone receptor-positive, early-stage breast cancer. Plasma estrogen levels are significantly lower in women who receive anastrozole compared with those who receive tamoxifen. We hypothesized, therefore, that anastrozole would have a more profound effect on cognitive function than tamoxifen, a mixed estrogen agonist/antagonist. To test this hypothesis we compared cognitive function in women with early-stage breast cancer who received tamoxifen with those who received anastrozole therapy in a cross-sectional study. We evaluated cognitive function, depression, anxiety, and fatigue in 31 postmenopausal women with early-stage breast cancer who were between the ages of 21 and 65 years and treated with tamoxifen or anastrozole for a minimum of 3 months. The results showed that women who received anastrozole had poorer verbal and visual learning and memory than women who received tamoxifen. Additional, prospective studies are needed to validate and confirm the changes in cognitive function associated with hormone therapy for breast cancer.

Pharmacokinetics and tolerability of exemestane in combination with raloxifene in postmenopausal women with a history of breast cancer

Raloxifene is a second-generation selective estrogen receptor modulator that reduces the incidence of breast cancer in postmenopausal women. Exemestane, a steroidal aromatase inhibitor, decreases contralateral new breast cancers in postmenopausal women when taken in the adjuvant setting. Preclinical evidence suggests a rationale for coadministration of these agents to achieve complete estrogen blockade. We tested the safety and tolerability of combination exemestane and raloxifene in 11 postmenopausal women with a history of hormone receptor-negative breast cancer. Patients were randomized to either raloxifene (60 mg PO daily) or exemestane (25 mg PO daily) for 2 weeks. Patients then initiated combination therapy at the same dose levels for a minimum of 1 year. Pharmacokinetic and pharmacodynamic data for plasma estrogens, raloxifene, exemestane, and their metabolites were collected at the end of single-agent therapy and during combination therapy. Plasma concentration-time profiles for each drug were unchanged with monotherapy versus combination therapy. Raloxifene did not affect plasma estrogen levels. Plasma estrogen concentrations were suppressed below the lower limit of detection by exemestane as monotherapy and when administered in combination with raloxifene. The most common adverse events of any grade included arthralgias, hot flashes, vaginal dryness and myalgias. In this small study, coadministration of raloxifene and exemestane did not affect the pharmacokinetics or pharmacodynamics of either agent to a significant degree in postmenopausal women. The combination of estrogen receptor blockade and suppression of estrogen synthesis is well tolerated and warrants further investigation.

Blockade of Estrogen Receptors Decreases CNS and Pituitary Prostaglandin Synthase Expression in Fetal Sheep

Background/Aims: Both prostaglandin E₂ (PGE₂) and estradiol stimulate fetal ACTH secretion and augment fetal ACTH responses to stress. We have reported that estradiol increases prostaglandin endoperoxide synthase-2 (PGHS-2), and we have proposed that there is a positive feedback relationship between estrogen and fetal hypothalamus-pituitary-adrenal (HPA) axis activity that is dependent upon PGHS activity in the fetal brain. The present study was designed to test the hypothesis that blockade of estrogen receptors in the fetal brain decrease PGHS-2 expression and reduces fetal HPA axis activity. Methods: In study 1, six time-dated pregnant ewes with chronically-catheterized twin fetuses were used. In each pregnancy, one twin was treated intracerebroventricularly (icv) with the estrogen receptor antagonist ICI 182,780 (25 µg/day; n = 6) while the other twin served as an age-matched control. In study 2, plasma samples were drawn from 10 singleton chronically-catheterized fetuses on alternating days until the time of spontaneous parturition. Results: ICI infusion caused significantly decreased PGHS-2 mRNA abundance in fetal central nervous system and pituitary, with the greatest decreases occurring in hippocampus and pituitary. There were no statistically significant changes in PGHS-1 mRNA. ICI infusion did not significantly change fetal plasma concentrations of pro-opiomelanocortin (POMC), ACTH, or cortisol in fetuses 130–134 days ges- tation (study 1) but did decrease the preparturient rise in plasma pro-opiomelanocortin concentrations in study 2. Conclusion: We conclude that PGHS-2 expression in the late-gestation fetal brain is in part stimulated by circulating estrogens in fetal plasma. Blockade of CNS estrogen receptors reduces preparturient plasma concentrations of POMC, but does not reduce fetal HPA axis activity in 130–134 day fetal sheep.

Update on the use of letrozole in breast cancer

Endocrine therapy is the mainstay of adjuvant treatment for hormonereceptor-positive early breast cancer. Letrozole is a potent third-generation aromatase inhibitor that suppresses plasma estrogen levels to near-undetectable levels in postmenopausal women. The results of well-controlled clinical trials have demonstrated the efficacy of letrozole over the gold-standard treatment, tamoxifen, in the neoadjuvant and upfront adjuvant settings and over placebo in the extended adjuvant setting (i.e., following 5 years of adjuvant tamoxifen). Important benefits in disease-free survival and especially distant disease-free survival have been demonstrated, in both low- and high-risk subgroups of patients (e.g., node-positive disease, prior chemotherapy). Both the efficacy and safety of letrozole for the adjuvant treatment of breast cancer are reviewed.

Ontogenic and nutritional regulation of steroid receptor and IGF-I transcript abundance in the prepubertal heifer mammary gland

In prepubertal cattle, mammary development is characterized by the growth of an epithelial-rich parenchyma (PAR) into the mammary fat pad (MFP). This proliferation and accumulation of mammary epithelial cells require estrogen. Paradoxically, both epithelial cell proliferation and PAR accumulation rate decline with rising plasma estrogen as puberty approaches. The possibility that variation in abundance of estrogen receptors (ERs) in PAR or MFP accounts for a portion of these effects has not been examined in cattle. Additionally, we recently demonstrated that MFP is highly responsive to exogenous estrogen, suggesting that this tissue may play a role in coordinating estrogen's effects on PAR; however, the developing bovine MFP has yet to be studied in detail. To address these hypotheses, Holstein heifers were assigned to planes of nutrition supporting body growth rates of 950 (E) or 650 (R) g/day and harvested every 50 kg from 100 to 350 kg body weight (BW). Post-harvest, their mammary glands were dissected into PAR and MFP compartments. Transcript abundance of genes encoding members of the ER family (ERalpha, ERbeta, and estrogen-related receptor alpha-1 (ERRalpha)) and estrogen-responsive genes (IGF-I and progesterone receptor (PR)) were measured in both mammary compartments by quantitative real-time RT-PCR. Significant expression was detected for all genes in both compartments, with the exception of the ERbeta gene. Transcript abundance of both ERalpha and IGF-I decreased linearly with increasing BW within both compartments. ERRalpha and PR expressions decreased with increasing BW in PAR but not in MFP. Nutrition stimulated ERalpha and ERRalpha expression in the PAR but had no effect on IGF-I or PR in either PAR or MFP. Overall, ERalpha and IGF-I transcript abundance are consistent with the drop in mammary epithelial cell proliferation and PAR accretion observed over development, but do not support a negative effect of nutrition on PAR growth.

Aromatase inhibitors: Assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.

Estrogen and prostate cancer: An eclipsed truth in an androgen‐dominated scenario

Prostate cancer is the commonest non-skin cancer in men. Incidence and mortality rates of this tumor vary strikingly throughout the world. Although several factors have been implicated to explain this remarkable variation, lifestyle and dietary factors may play a dominant role, with sex hormones behaving as intermediaries between exogenous factors and molecular targets in development and progression of prostate cancer. Human prostate cancer is generally considered a paradigm of androgen-dependent tumor; however, estrogen role in both normal and malignant prostate appears to be equally important. The association between plasma androgens and prostate cancer remains contradictory and mostly not compatible with the androgen hypothesis. Similar evidence apply to estrogens, although the ratio of androgen to estrogen in plasma declines with age. Apart from methodological problems, a major issue is to what extent circulating hormones can be considered representative of their intraprostatic levels. Both nontumoral and malignant human prostate tissues and cells are endowed with key enzymes of steroid metabolism, including 17betahydroxysteroid dehydrogenase (17betaHSD), 5beta-reductase, 3alpha/3betaHSD, and aromatase. A divergent expression and/or activity of these enzymes may eventually lead to a differential prostate accumulation of steroid derivatives having distinct biological activities, as it occurs for hydroxylated estrogens in the human breast. Locally produced or metabolically transformed estrogens may differently affect proliferative activity of prostate cancer cells. Aberrant aromatase expression and activity has been reported in prostate tumor tissues and cells, implying that androgen aromatization to estrogens may play a role in prostate carcinogenesis or tumor progression. Interestingly, many genes encoding for steroid enzymes are polymorphic, although only a few studies have supported their relation with risk of prostate cancer. In animal model systems estrogens, combined with androgens, appear to be required for the malignant transformation of prostate epithelial cells. Although the mechanisms underlying the hormonal induction of prostate cancer in experimental animals remain uncertain, there is however evidence to support the assumption that long term administration of androgens and estrogens results in an estrogenic milieu in rat prostates and in the ensuing development of dysplasia and cancer. Both androgen and estrogen have been reported to stimulate proliferation of cultured prostate cancer cells, primarily through receptor-mediated effects. As for estrogens, the two major receptor types, ERalpha and ERbeta, are expressed in both normal and diseased human prostate, though with a different cellular localization. Since these two receptors are different in terms of ligand binding, heterodimerization, transactivation, and estrogen response element activity, it is likely that an imbalance of their expression may be critical to determine the ultimate estrogen effects on prostate cancer cells. In prostate cancer, ERbeta activation appears to limit cell proliferation directly or through ERalpha inhibition, and loss of ERbeta has been consistently associated with tumor progression. Several splicing variants of both ERalpha and ERbeta exist. Little is known about their expression and function in the human prostate, although reciprocal regulation and interaction with gene promoter both warrant further investigation. In summary, although multiple consistent evidence suggests that estrogens are critical players in human prostate cancer, their role has been only recently reconsidered, being eclipsed for years by an androgen-dominated interest.

MP-17.07: Endogenously expressed sex hormone-binding globulin (SHBG) mediates the steroid response of prostate cancer cells

Sex hormone-binding globulin (SHBG), a protein that binds plasma androgens and estrogens, participates in the initial steps of a membrane-based steroid signaling pathway in human prostate cells that involves a specific receptor, RSHBG, and is independent of the androgen receptor (AR). We have recently shown that SHBG is locally expressed in prostate cells. We hypothesized that locally expressed SHBG has at least two functions in prostate cells, 1) to regulate membrane based steroid signalling, and 2) to bind androgens intracellularly, thereby regulating AR activity.

Effect of Estrogen on Leptin and Expression of Leptin Receptor Transcripts in Prepubertal Dairy Heifers

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17β-estradiol for 3 consecutive days. Neither ovarian status nor 17β-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-RTOTAL). Ob-RTOTAL was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-RTOTAL in the hypothalamus, but averaged less than 3% of Ob-RTOTAL in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-RTOTAL in the hypothalamus and for nearly all of Ob-RTOTAL in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17β-estradiol on Ob-R expression in selected tissues. Treatment with 17β-estradiol reduced Ob-RTOTAL, Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17β-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.

Effects of steroidal and nonsteroidal aromatase inhibitors on markers of bone turnover in healthy postmenopausal women

IntroductionIn contrast to nonsteroidal aromatase inhibitors, the steroidal aromatase inactivator exemestane does not have detrimental effects on bone in animal models. This study was designed to compare the effects of exemestane with the nonsteroidal aromatase inhibitors anastrozole and letrozole on serum and urine levels of biomarkers of bone turnover in healthy postmenopausal women.MethodsChanges in the concentrations of bone-turnover markers, estrogens, and lipids were assessed after daily administration of exemestane (25 mg), letrozole (2.5 mg), anastrozole (1 mg), or placebo for 24 weeks in healthy postmenopausal women. The primary end point was the percentage change from baseline in bone-turnover-marker levels at week 24. The baseline-adjusted area under the curve (AUC) for weeks 0–12 and 0–24 was calculated to evaluate changes in bone turnover over time, rather than at discrete time points.ResultsSeventy-four (88%) of 84 randomized subjects were evaluable for bone-marker assays. Reductions in plasma estrogen levels and increases in bone-resorption markers were comparable for each aromatase inhibitor. Uniquely, exemestane consistently increased the percentage change from baseline in the level of serum procollagen type I N-terminal propeptide (PINP), a marker of bone formation, at week 24. In the active-treatment groups, the baseline-adjusted AUC at weeks 0–12 and 0–24 for PINP was significantly greater for exemestane than the other aromatase inhibitors.ConclusionExemestane increased serum levels of the bone-formation marker PINP after 24 weeks, suggesting a specific bone-formation effect related to its androgenic structure. Potential effects on cortical bone and reduced fracture risk must be verified in a comparative clinical trial.

Female Gender-specific Inhibition of KCNQ1 Channels and Chloride Secretion by 17β-Estradiol in Rat Distal Colonic Crypts

The estrogen sex steroid 17beta-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl(-) secretion in the female rat distal colonic crypt by the inhibition of basolateral K(+) channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K(+) current was shown to be attenuated by pretreatment with rottlerin, a PKCdelta-specific inhibitor. In whole cell patch-clamp analysis, 17beta-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCdelta in comparison to male tissue. PKCdelta and PKA were activated at 5 min in response to 17beta-estradiol in female distal colonic crypts only. Both PKCdelta- and PKA-associated with the KCNQ1 channel in response to 17beta-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCdelta as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCdelta- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.

Prospective study of grapefruit intake and risk of breast cancer in postmenopausal women: the Multiethnic Cohort Study

In vitro and in vivo studies have shown that cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of oestrogens. There is evidence that grapefruit, an inhibitor of CYP3A4, increases plasma oestrogen concentrations. Since it is well established that oestrogen is associated with breast cancer risk, it is plausible that regular intake of grapefruit would increase a woman's risk of breast cancer. We investigated the association of grapefruit intake with breast cancer risk in the Hawaii–Los Angeles Multiethnic Cohort Study, a prospective cohort that includes over 50 000 postmenopausal women from five racial/ethnic groups. A total of 1657 incident breast cancer cases were available for analysis. Grapefruit intake was significantly associated with an increased risk of breast cancer (relative risk=1.30, 95% confidence interval 1.06–1.58) for subjects in the highest category of intake, that is, one-quarter grapefruit or more per day, compared to non-consumers (Ptrend=0.015). An increased risk of similar magnitude was seen in users of oestrogen therapy, users of oestrogen+progestin therapy, and among never users of hormone therapy. Grapefruit intake may increase the risk of breast cancer among postmenopausal women.

SPATIAL AND TEMPORAL REGULATION OF BMP-2, -6, -15 AND GDF-9 mRNA EXPRESSION IN DEVELOPING PORCINE OVARIAN FOLLICLES

Members of the transforming growth factor beta (TGF-beta) superfamily, namely the bone morphogenetic proteins (BMPs) and growth differentiation factor 9 (GDF-9), have been shown to play important roles during follicular development and ovulation. However, despite the wealth of information available in rodent and ruminant species, very little is known about the role of these genes in the porcine ovary. Therefore, the aims of the present study were to investigate the expression of BMP-2, -6, -15 and GDF-9 mRNA in the oocyte, granulosa cells and theca cells of porcine follicles and to determine if changes occur in the levels of expression of these genes during follicular growth. Oocyte, granulosa cells and theca cells were recovered from antral follicles (2-10 mm in diameter) of first parity sows at day 1, 2 and 4 after weaning or at day 14, 16 and 20 of their subsequent estrous cycle. These time points correspond to specific phases of follicular growth; the recruitment of a growing cohort of follicles, the mid-selection phase and the final selection of the pre-ovulatory follicle population. Plasma estrogen and progesterone analysis was used to confirm the expected physiological status of each animal. Follicular fluid estradiol concentration in day 4 and day 20 animals was also used to determine whether the follicles had been exposed to the LH surge. Total RNA was extracted, reverse transcribed and the levels of expression for these genes were determined using real-time PCR. The delta Ct values were calculated using cyclophilin as the internal control and the data were analysed using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC). It is particularly interesting to notice that both BMP-2 and BMP-15 were expressed in all three cell types but were expressed at considerably higher levels in the oocyte (p < 0.001). BMP-2 mRNA levels increased in granulosa cells (P = 0.018) and theca cells (P < 0.001) from the recruitment phase to the mid-selection phase, remained constant until the final selection phase and decreased after the LH surge. BMP-15 mRNA levels also increased in both cell types from the recruitment phase to the mid-selection phase, however the levels decreased in the granulosa cells of the final pre-ovulatory follicle population while remaining constant during this phase in the theca cells (P < 0.05). As opposed to BMP-2, the level of BMP-15 mRNA did not change in response to the LH surge. Finally, BMP-15 mRNA levels showed a strong tendency to decrease in the oocytes from recruitment to the final selection phase (P = 0.054). These results confirm that the oocyte is the main source of BMP-2 and BMP-15 but contrary to other studies, their expression was also found in granulosa cells and theca cells. These differences suggest that follicular growth and development may be regulated differently in swine. (poster)

Fulvestrant (Faslodex®)—How to Make a Good Drug Better

Fulvestrant (Faslodex); AstraZeneca Pharmaceuticals, Wilmington, DE) is an estrogen receptor (ER) antagonist with a novel mode of action; it binds, blocks, and increases degradation of ER. Fulvestrant (at the approved dose [250 mg/month]) is at least as effective as anastrozole (1 mg/day) in the treatment of postmenopausal women with hormone receptor-positive advanced breast cancer (HR(+) ABC) progressing or recurring on antiestrogen therapy, and is also an active first-line treatment. Although fulvestrant (250 mg/month) is clearly effective, it takes 3-6 months to achieve steady-state plasma levels. Steady-state concentrations are approximately twofold higher than those achieved with a single dose; reaching this earlier, for example, via a loading-dose (LD) regimen (250 mg/month plus 500 mg on day 0 and 250 mg on day 14 of month 1), may allow responses to be achieved more quickly and limit the possibility of early relapse. Fulvestrant high-dose (HD) regimens (500 mg/month) offer the possibility of greater antitumor activity, because (a) ER downregulation is a dose-dependent process (an approximately 70% reduction is observed with a single 250 mg dose of fulvestrant) and (b) evidence correlates greater ER downregulation with superior efficacy. A fulvestrant HD regimen offers the potential of achieving near 100% ER downregulation. There is also potential to increase fulvestrant-ER binding by reducing plasma estrogen levels, for example, with concomitant aromatase inhibitor treatment. Several ongoing trials use LD, HD, and combination regimens; results from these studies are awaited with interest. Meanwhile, fulvestrant (250 mg/month) remains a valuable additional endocrine treatment for postmenopausal women with HR(+) ABC recurring or progressing on antiestrogen therapy.

Structural and metabolic status of erythrocytes in patients with juvenile uterinebleedings

Study results of investigation of peripheral erythron component in patients with juvenile uterine bleedings. We found marked changes of metabolic and morphologic status of erythrocytes: decreased mean mass of their dry weight and increased number of their transformed and degenerative forms. Cytochemical studies registered decreased content of sulfa radicals and lipoproteins in cytomembranes of erythrocytes. The most marked change of peripheral erythron component was seen in yuvenile uterine bleedings which are ongoing in the setting of high plasma estrogen level.

Imidazolylmethylbenzophenones as Highly Potent Aromatase Inhibitors

Suppression of tumor and plasma estrogen levels by inhibition of aromatase is one of the most effective treatments for postmenopausal breast cancer patients. Starting from an easy, synthetically accessible, benzophenone scaffold, a new class of potent aromatase inhibitors was synthesized, endowed with high selectivity with respect to 17 alpha-hydroxylase/17,20-lyase (CYP17). Compounds 1b and 1d proved to be among the most potent inhibitors described so far.

Influence of letrozole on plasma estrogen levels in postmenopausal patients with metastatic breast cancer and the effect of estrogen levels on response rates

1078 Background: The aromatase inhibitor (AI) letrozole and other members of this treatment group are very effective in the treatment of postmenopausal women with breast cancer. AIs markedly reduce estrogen levels in postmenopausal women by inhibiting aromatase. At menopause estradiol levels are about 25 pmol/l. Estradiol levels are higher in plasma of women with breast cancer as compared with women with no breast cancer. The purpose of this prospective study is to evaluate estradiol levels before and during letrozole treatment and the effect of primary estradiol levels and their decrease on response rate. Methods: Letrozole 2.5 mg/day orally was given to 50 patients (pts) with metastatic and locally advanced breast cancer (MBC). All pts had measurable disease. Overnight fasting clotted blood samples used for assessment of serum levels of estradiol at baseline and after 3, 6 and 12 months. Estradiol levels were compared to clinical response in this single stage study. Results: Mean age was 56 years (range 46–89 y). Mean years from menopause to starting letrozole was 9 years.12% of pts received previous hormonotherapy and and 30% received chemotherapy for their metastatic disease. Estradiol levels decreased from a mean of 40 pmol/l before letrozole to 40 pmol/l and &lt;40 pmol/l in 19 pts (38%). No difference in estradiol depression rate was found between the 2 groups. In the first group (estradiol &gt;40 pmol/l) objective response rate was 48%: 4 complete responses (CR) and 11 partial responses (PR) out of 31 pts, 12 pts had stabilization of the disease (SD). In the second group 3 pts had CR, 5 had PR: objective response rate of 42% (8/19), and 42% SD. There is a trend (not significant) for higher response rate in pts with higher estradiol levels at the beginning of treatment. Conclusions: Letrozole was found to cause very high estradiol suppression and high response rates in postmenopausal women with advanced breast cancer. A trend was found for higher response rates in pts with higher estradiol levels at baseline although difference was not significant. This is the first study to address this issue. No significant financial relationships to disclose.