Treatment with high-dose estrogen (diethylstilbestrol) significantly decreases plasma estrogen and androgen levels but does not influence in vivo aromatization in postmenopausal breast cancer patients

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E₂) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n = 9) and in vivo aromatization (n = 3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5 mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P < 0.05 for all). While we found no change in total body aromatization and plasma estrone (E₁) levels, estradiol (E₂) and estrone sulfate (E₁S) were suppressed by a mean of 48.8 and 68.2%, respectively (P = 0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P < 0.05 for all androgens). In contrast, no change in plasma progesterone or 17-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.     

Beneficial effects of weight loss on plasma apolipoproteins in postmenopausal women

A total of 39 postmenopausal women 40–70 years of age and undergoing hormone replacement therapy participated in a 6-month weight reduction program, which consisted of a low calorie diet (5040 KJ/day) and phentermine hydrochloride therapy. Subjects had an average body mass index of 35.95±5.32 kg/m² and 42.20±11.0 kg of total fat. Body mass index, plasma lipids, total and trunk fat, and plasma apoproteins were measured at baseline and after 3 and 6 months of the weight reduction program. Subjects experienced an overall 10% weight loss during the treatment period (P<0.001). Plasma LDL cholesterol and triglycerides were reduced by 18% and 15% (P<0.01) respectively, whereas HDL cholesterol was increased by 9% (P<0.01) over the 6-month period. Plasma apoproteins were significantly affected by weight loss. Plasma apolipoprotein (apo) B concentrations were reduced 6.5% (P<0.01), and apo C-III and apo E were reduced by 9% over 6 months (P<0.01). The observed decreases in plasma apo B were significantly correlated with the observed changes in plasma cholesterol (r=0.356, P<0.01) over 3 months. In addition, changes in plasma triglycerides were correlated with changes in both apo C-III (r=0.436) and apo E (r=0.354) over 6 months. These results suggest that weight loss may have multifactorial effects on lipoprotein metabolism, resulting in better plasma lipid and apoprotein profiles.     

Effects of long-term treatment with estradiol or clomiphene citrate on bone maintenance, and pituitary and uterine weights in ovariectomized rats

Long-term estrogen replacement therapy in postmenopausal women can bring relief to hot flushes and reduce loss of bone mass due to osteoporosis, however, such treatment often can cause uterine hyperplasia and other undesirable effects. This study compared changes in bone mineral content (BMC), uterine weight, pituitary weight and pituitary gonadotropin content in the ovariectomized rat model following treatment with estradiol (E₂) or two levels of clomiphene citrate (CC), an estrogen agonist/antagonist.

Groups (n = 8–12) of adult ovariectomized (OVX) rats were implanted with E₂ pellets (5 μg/day) or injected subcutaneously with CC at 1 mg/kg body wt (CC-1) or 5 mg/kg body wt (CC-5) twice weekly for 12 months. Placebo implanted OVX and intact (INT) female rats served as negative and positive controls, respectively. Following treatment, the uterus, pituitary gland and right femur were collected from each animal.

E₂ treatment increased (P<0.05) uterine weight compared to all other treatment groups, while both CC doses increased uterine weight over the OVX group only (E₂, 0.24±0.03; INT, 0.14±0.01; CC-1, 0.06±0.01; CC-5, 0.07±0.01; and OVX, 0.02±0.01 g per 100 g body wt). Pituitary weight was increased 15-fold (P<0.05) by E₂ treatment over all other treatment groups (E₂, 65.7±13.9; INT, 4.0±0.5; CC-1, 3.3±0.03; CC-5, 2.7±0.2; and OVX, 2.9±0.02 mg per 100 g body wt). Both E₂ and CC treatments reduced pituitary luteinizing hormone and follicle stimulating hormone content (μg/pit) to INT levels and were lower (P<0.05) than OVX levels. Mean BMC of E₂, CC-1- or CC-5-treated rats was greater (P<0.05) than that of either the INT or OVX groups, while INT animals had a higher BMC compared to OVX animals (E₂, 0.027±0.003; CC-1, 0.026±0.001; CC-5, 0.028±0.001; INT, 0.021±0.001; and OVX, 0.017±0.001 g/cm per 100 g body wt). These data indicate that CC has the potential to reduce bone mineral loss without causing other undesirable effects, including uterine hyperstimulation, and thus needs to be further investigated.     

Relationship between aromatase activity and steroid receptor levels in ovarian tumors from postmenopausal women

Aromatase activity, as well as steroid receptors, exists in nonfunctional ovarian tumors. Steroid receptor status has been reported to be related to prognosis in ovarian cancer patients. We determined aromatase activity and progesterone receptor (PR) and estrogen receptor (ER) levels in 43 ovarian tumors obtained from postmenopausal women. Aromatase activity was detected in 35 tumors (81%), PR in 21 tumors (49%) and ER in 13 tumors (30%). Eighty-three percent (10/12) of mucinous cystadenoma tissues showed positive PR with high aromatase activity, while 93% (13/14) of malignant tumors showed negative PR and low aromatase activity. Aromatase activity was detected in 95% (20/21) of PR-positive tumors, being greater than in PR-negative tumors (P < 0.002). There was a positive correlation between aromatase activity and PR (r_s = 0.49, P < 0.001). However, there was no correlation between aromatase activity and ER. In 17 patients (43%), the serum estradiol level was higher than 30 pg/ml and there was a positive correlation among estradiol, estrone, androstenedione and testosterone. However, serum steroid levels were not correlated with aromatase activity, PR or ER. Aminoglutethimide inhibited aromatase activity of benign and malignant ovarian tumors, uterine myoma, choriocarcinoma cells and purified human placental P-450arom in a similar manner. These results suggest that aromatase activity is correlated with PR in ovarian tumors of postmenopausal women. In addition to steroid receptor status, aromatase activity may be a useful prognostic factor in ovarian cancers.     

Influence of tamoxifen, aminoglutethimide and goserelin on human plasma IGF-I levels in breast cancer patients

Plasma insulin-like growth factor-I (IGF-I) was measured in breast cancer patients before and during treatment with tamoxifen, goserelin or aminoglutethimide. 24 out of 27 postmenopausal women treated with tamoxifen 20 or 30 mg daily experienced a decrease in plasma IGF-I levels (mean levels before treatment 14.8 nM, during treatment 10.2 nM, P < 0.001). In 8 out of 12 premenopausal breast cancer patients there was a reduction in plasma IGF-I during treatment with goserelin (mean levels before treatment 23.3 nM, during treatment 19.4 nM, P = 0.052). Contrary, 15 out of 17 postmenopausal women treated with the aromatase inhibitor aminoglutethimide had an increase in plasma IGF-I level (mean level before treatment 17.0 nM, during treatment 21.1 nM, P < 0.01). These preliminary results indicate that different forms of endocrine treatment of breast cancer may influence plasma IGF-I levels in different directions.     

Aromatase activity, serum oestradiol and their correlation with demographic indices

Peripheral aromatase activity was measured in 24 postmenopausal women suffering from advanced breast cancer. The % conversion of androstenedione to oestrone was then assessed for a significant correlation with age, weight, height, Quetelets index (weight/height², Q.I.) and length of menopause. Serum oestradiol (E₂) levels were measured in 22 of the subjects and compared with the same indices. There was no correlation between E₂ or aromatase activity with the length of menopause (P = 0.3 and P = 0.5, respectively). In our data aromatase activity did not correlate with age (P > 0.5, N = 22). Serum E₂ levels (P = 0.07, N = 20) expressed a negative correlation (i.e. decreased) with age. There was also a poor correlation between aromatase activity and weight of Quetelets index (P = 0.3, N = 20 for both). Serum E₂ levels showed a statistically significant correlation with weight (P = 0.01, N = 21), but the relationship with Quetelets index just failed to attain statistical significance (P = 0.07, N = 20). In both cases the regression line was positive. When aromatase activity was correlated with serum E₂ levels the regression line was positive but not statistically significant (P = 0.4, N = 22). The data indicate that aromatase activity is only one factor determining the differences in serum E₂ levels between postmenopausal women.     

The influence of dietary vitamin E, fat, and methionine on blood cholesterol profile, homocysteine levels, and oxidizability of low density lipoprotein in the gerbil

A 90-day feeding study with gerbils was conducted to evaluate the influence of dietary vitamin E levels (25 mg/kg diet, 75 mg/kg, 300 mg/kg, and 900 mg/kg), two levels of dietary methionione (casein or casein+L-methionine (1% w/w)) and two sources of lipid (soybean oil [20%] or soybean oil [4%]+coconut oil [16%, 1:4 w/w]) upon serum lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol). In addition, this study examined the effects of diet-induced hyperhomocysteinemia and supplemental dietary vitamin E on the oxidation of low density lipoproteins. Tissue vitamin E (heart, liver, and plasma) demonstrated a dose response (P≤0.001) following the supplementation with increasing dietary vitamin E (25, 75, 300, and 900 mg/kg). In addition, tissue vitamin E levels were found to be higher (P≤0.001) in those animals receiving a combination of coconut oil+soybean oil as compared to the group receiving soybean oil solely. Blood cholesterol profiles indicated an increase (P≤0.001) in total cholesterol and LDL cholesterol by the influence of saturated fat and supplemental methionine. Low-density lipoprotein cholesterol profile demonstrated a reduction (P≤0.001) at the higher dietary vitamin E levels (300 and 900 mg/kg) as compared to the 25 mg/kg and 75 mg/kg dietary vitamin E. Plasma protein carbonyls were not influenced by dietary vitamin E nor by supplemental methionine intake. In vitro oxidation of LDL showed that vitamin E delayed the lag time of the oxidation phase (P≤0.001) and reduced total diene production (P≤0.001). On the contrary, supplemental methionine decreased (P≤0.001) the delay time of the lag phase, whereas total diene production was increased (P≤0.001). Plasma lipid hydroperoxides were significantly reduced (P≤0.05) with supplemental dietary vitamin E, whereas supplemental L-methionine (1%) resulted in a significant (P≤0.05) increase in lipid plasma hydroperoxide formation. Plasma homocysteine was elevated (P≤0.001) with supplemental dietary L-methionine (1%) as well as the inclusion of dietary saturated fat. The present data showed that 1) a combination of dietary lipids (saturated and unsaturated fatty acids) as well as vitamin E and methionine supplementation altered blood cholesterol lipoprotein profiles; 2) in vitro oxidation parameters including LDL (lag time and diene production) and plasma hydroperoxide formations were affected by vitamin E and methionine supplementation; and 3) plasma homocysteine concentrations were influenced by supplemental methionine and the inclusion of dietary saturated fat.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

Studies on the changes in plasma lipids and lipoproteins in patients with benign and malignant breast cancer

Experimental studies and epidemiological data suggest that, high fat diet increases the risk of developing breast cancer, both in animal and in human population. Cases of postmenopausal, untreated women with malignant and benign breast tumors, were compared for their age, body weight, plasma lipid fractions and lipoproteins. There was a significant increase in body weight, total plasma lipids, total cholesterol, LDL--cholesterol, VLDL--cholesterol, phospholipids, triacylglycerols, free fatty acids in malignant breast cancer patients. HDL--cholesterol had been significantly decreased in benign and malignant patients when compared with the control subjects.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Relationship between indices of iron status and coronary risk factors including diabetes and the metabolic syndrome in Saudi subjects without overt coronary disease

There have been inconsistent reports on the relationship between iron status and coronary artery diseases (CAD), and little data on this relationship in non-Caucasian populations.

We assessed dietary iron by questionnaire and measured serum iron and ferritin levels in 270 Saudi male subjects without established CAD, 130 of whom were angiogram negative. Serum lipid profile, glucose, high sensitivity-C reactive protein (hs-CRP), serum soluble intercellular adhesion molecules-1 (sICAM-1), and caeruloplasmin were measured in all subjects.

The angiogram negative patients, had lower serum ferritin (p<0.05) and iron (p<0.0001) levels than the 140 subjects without reported cardiovascular diseases (CVD). Serum iron correlated with serum triglycerides (p<0.0001) and total cholesterol (p<0.05) levels for this latter group and the groups combined. Serum ferritin correlated with serum total cholesterol and low-density lipoprotein (LDL)-cholesterol in the combined group (p<0.05), and was correlated with blood glucose and serum LDL-cholesterol (p<0.05) in the subjects without reported CVD. After adjustment for confounding variables, serum iron levels remained a significant correlate with total calorie intake and serum triglycerides. Serum ferritin also correlated significantly with cholesterol intake and fasting serum total cholesterol. Dietary iron was significantly related to dietary cholesterol and fiber, age, smoking habits, and serum total cholesterol level.

Hence, indices of iron status were related to several coronary risk factors in the Saudi population.     

Lipid and lipoprotein profile in menopausal transition. Effects of hormones, age and fat distribution.

The behavior of lipoproteins during the menopausal transition and their relationship with sex hormones and body fat distribution is still unclear. Our aim was to evaluate atherogenic IDL, LDL, Lp(a) and antiatherogenic HDL lipoproteins in four groups of women: premenopausal (n = 20), menopausal transition women with menstrual bleeding (n = 31), menopausal transition women with 3 to 6 months amenorrhea (n = 36), and postmenopausal women (n = 30). We also measured their FSH, LH and estradiol levels along with BMI and waist circumference. Menopausal transition and postmenopausal women showed higher values of waist circumference (p < 0.0032), LDL-cholesterol (p < 0.002), IDL-cholesterol (p < 0.002) and apoprotein B (p < 0.0001) than premenopausal women. Total-cholesterol (p < 0.0001), triglycerides (p < 0.004), IDL-cholesterol and Lp(a) were higher in menopausal transition women with amenorrhea and in postmenopausal women in comparison with premenopausal women. After adjustment according to age and waist circumference, multiple regression analysis showed the increase in total-cholesterol and LDL-cholesterol to be linearly associated to menopausal status and estradiol concentration, whereas Lp(a) was only related to menopausal status. Age was found to be an independent variable in relation to apoprotein B concentration changes. The effect of menopausal status on TG levels did not remain in the model when age, waist and BMI were included (beta = 0.05, p = 0.356). HDL-cholesterol levels were the same in all the groups. Menopause, age and the increase in abdominal fat distribution were three independent and significant factors impairing lipoprotein profiles from the beginning of the menopausal transition.     

Effect of Neoadjuvant Chemotherapy on the Serum Levels of Bone Turnover Markers in Women with Early-Stage Breast Cancer

Background

To evaluate effects of neoadjuvant chemotherapy on the bone turnover markers of preoperational breast cancer patients.

Methods

Forty-one breast cancer patients (29 premenopausal and 12 postmenopausal) and 60 healthy women (30 premenopausal and 30 postmenopausal) aged 30-64 years, were evaluated for their bone status. Serum levels of the bone formation markers PINP and BAP, as well as the resorption markers ICTP and β-Crosslaps in addition to E2, FSH, 25(OH)D and PTH were measured at the initial diagnosis and at 24 hours after each four chemotherapy cycles. BMD T-scores were determined in 12 patients 6 months after the neoadjuvant chemotherapies.

Results

The baseline levels of both bone formation and resorption markers in premenopausal patients were higher than in premenopausal healthy women (p<0.05), while no statistic difference was observed between postmenopausal patients and postmenopausal healthy women. Regardless of the menopausal status, chemotherapy increased the ICTP and β-Crosslaps levels (p<0.05), but decreased the BAP and PINP levels (p<0.05), the later one significantly more with Taxane medication (p<0.01, p<0.05). Chemotherapy caused significant decreases of 25(OH)D levels in premenopausal (p<0.01) and postmenopausal (p<0.05) patients, however, did not affect the PTH concentrations. In premenopausal patients the E2 level decreased, while the FSH level increased after chemotherapy (p<0.05). Patients with pronounced ICTP and β-Crosslaps combined with reduced BAP and PINP serum concentrations after neoadjuvant chemotherapies were prone to develop osteoporosis 6 month later.

Conclusions

Neoadjuvant chemotherapy appeared to promote bone resorption and inhibit bone formation in both postmenopausal and premenopausal early-stage breast patients.     

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E₂), estrone (E₁) and E₁S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E₁ levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E₁ cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E₂ concentration in ER positive tumours versus normal tissue (p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E₂ concentrations in ER− tumours (p < 0.01 and <0.001 comparing E₂ levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E₂ in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.     

Serum Lipids and Breast Cancer Risk: A Meta-Analysis of Prospective Cohort Studies

Purpose

Epidemiologic studies exploring causal associations between serum lipids and breast cancer risk have reported contradictory results. We conducted a meta-analysis of prospective cohort studies to evaluate these associations.

Methods

Relevant studies were identified by searching PubMed and EMBASE through April 2015. We included prospective cohort studies that reported relative risk (RR) estimates with 95% confidence intervals (CIs) for the associations of specific lipid components (i.e., total cholesterol [TC], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and triglycerides [TG]) with breast cancer risk. Either a fixed- or a random-effects model was used to calculate pooled RRs.

Results

Fifteen prospective cohort studies involving 1,189,635 participants and 23,369 breast cancer cases were included in the meta-analysis. The pooled RRs of breast cancer for the highest versus lowest categories were 0.96 (95% CI: 0.86–1.07) for TC, 0.92 (95% CI: 0.73–1.16) for HDL-C, 0.90 (95% CI: 0.77–1.06) for LDL-C, and 0.93 (95% CI: 0.86–1.00) for TG. Notably, for HDL-C, a significant reduction of breast cancer risk was observed among postmenopausal women (RR = 0.77, 95% CI: 0.64–0.93) but not among premenopausal women. Similar trends of the associations were observed in the dose-response analysis.

Conclusions

Our findings suggest that serum levels of TG but not TC and LDL-C may be inversely associated with breast cancer risk. Serum HDL-C may also protect against breast carcinogenesis among postmenopausal women.     

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 (E₁), estradiol (E₂) and estrone sulfate (E₁S) levels in the ultra-low range. Following incubation with [³H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E₁S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E₁, E₂) were separated by chromatography (LH-20). Estrone and E₁S (following hydrolysis) were converted into E₂, 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 E₁, E₂ and E₁S, 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 E₁, 0.67 pmol/L for E₂, and 0.55 pmol/L for E₁S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E₁, 5.1% for E₂ and 6.1% for E₁S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E₁, E₂, and E₁S, respectively. Considering normal plasma levels for E₂ (15 pmol/L), E₁ (80 pmol/L) and E₁S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E₂, E₁ and E₁S 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.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

Leptin expression in breast nipple aspirate fluid (NAF) and serum is influenced by body mass index (BMI) but not by the presence of breast cancer.

While obesity is a known risk factor for postmenopausal breast cancer, the molecular mechanisms involved are unclear. Systemic levels of leptin, the product of the ob (obesity) gene, are increased in obese individuals (body mass index, BMI, over 25) and are higher in women than men. Leptin has been found to stimulate the growth of breast cancer cells in vitro. Our goal was to determine whether leptin was 1) present in nipple aspirate fluid (NAF), and 2) whether NAF leptin levels were associated with a) levels in serum, b) obesity, and c) breast cancer. We collected and evaluated NAF specimens from 83 subjects and serum specimens from 49 subjects. NAF leptin was detectable in 16/41 (39 %) of premenopausal and 21/42 (50 %) postmenopausal subjects. NAF leptin was significantly lower (p = 0.042) in premenopausal than postmenopausal women with a BMI < 25, but not in those with a higher BMI. NAF leptin was significantly associated with BMI in premenopausal (p = 0.011) but not in postmenopausal women. Serum leptin was associated with BMI in both premenopausal and postmenopausal women (p = 0.0001 for both). NAF and serum leptin were associated in premenopausal (p = 0.02) but not postmenopausal women. Neither NAF nor serum leptin was associated with premenopausal or postmenopausal breast cancer. Our findings include that 1) leptin is present in the breast and detectable in a subset of NAF samples, 2) NAF leptin in premenopausal but not postmenopausal women parallels serum leptin levels, and 3) neither NAF nor serum levels of leptin were associated with premenopausal or postmenopausal breast cancer.     

Estrogens in tissues: uptake from the peripheral circulation or local production

Because intratissue levels of estrogens may be more important for the understanding of the endocrine status of an organ than are peripheral plasma levels, the concentrations of estrone and estradiol have been measured in normal and pathological breast and uterine specimens. Some breast tumors were collected in countries with differences in incidence and natural history of the disease. In other samples the subcellular distribution of the steroids was studied. Estrone levels did show much less variation than estradiol levels. Not related to estrogen receptors, estradiol levels were higher in uterine than in breast tissues. Also, the subcellular distribution observed could not be explained by changes in receptors. Malignant breast tumors of premenopausal and postmenopausal women contained similar amounts of estradiol. Unexplained large differences were found in the intratissue estradiol levels obtained in different countries.     

Effect of superovulation on maternal serum progesterone concentration, uterine and fetal weights at weeks 7 and 15 of pregnancy in Javanese Thin-Tail ewes

Twenty-one pregnant ewe lambs (nine superovulated and 12 non-superovulated) were used to study the effects of superovulation (injecting 700 IU PMSG at the end of diestrus) on maternal serum progesterone concentrations, uterine and fetal weights at weeks 7 and 15 of pregnancy. In the ewes sacrificed at week 7 of pregnancy, superovulation increased the mean number of corpora lutea (P<0.01), fetuses (P<0.01), maternal mean serum progesterone concentration (P<0.01), mean uterine weight (P<0.05), total fetal weight (P<0.01), and average fetal weight (P<0.01) by 133%, 69%, 354%, 66%, 150% and 40%, respectively, when compared to non-superovulated ewes. In the ewes sacrificed at week 15 of pregnancy, superovulation increased the number of corpora lutea (P<0.01), fetuses (P<0.05), maternal serum progesterone concentration (P<0.01), uterine weight (P<0.05), total fetal weight (P>0.05), and average fetal weight (P<0.05) by 207%, 20%, 84%, 37%, 29% and 24%, respectively, compared to those non-superovulated ewes. It was concluded that the increased number of corpora lutea and, therefore, their hormonal secretions by superovulation could increase uterine and fetal growth and development.