Relation of Serum Estrogen Metabolites with Terminal Duct Lobular Unit Involution Among Women Undergoing Diagnostic Image-Guided Breast Biopsy.

Higher levels of circulating estrogens and estrogen metabolites (EMs) have been associated with higher breast cancer risk. In breast tissues, reduced levels of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have also been linked to elevated breast cancer risk. However, it is unknown whether reduced TDLU involution mediates the risk associated with circulating EMs. In a cross-sectional analysis of 94 premenopausal and 92 postmenopausal women referred for clinical breast biopsy at an academic facility in Vermont, we examined the associations of 15 EMs, quantified using liquid chromatography-tandem mass spectrometry, with the number of TDLUs and acini count/TDLU using zero-inflated Poisson regression with a robust variance estimator and ordinal logistic regression models, respectively. All analyses were stratified by menopausal status and adjusted for potential confounders. Among premenopausal women, comparing the highest vs. the lowest tertiles, levels of unconjugated estradiol (risk ratio (RR) = 1.74, 95 % confidence interval (CI) = 1.06-2.87, p trend = 0.03), 2-hydroxyestrone (RR = 1.74, 95 % CI = 1.01-3.01, p trend = 0.04), and 4-hydroxyestrone (RR = 1.74, 95 % CI = 0.99-3.06, p trend = 0.04) were associated with significantly higher TDLU count. Among postmenopausal women, higher levels of estradiol (RR = 2.09, 95 % CI = 1.01-4.30, p trend = 0.04) and 16α-hydroxyestrone (RR = 2.27, 95 % CI = 1.29-3.99, p trend = 0.02) were significantly associated with higher TDLU count. Among postmenopausal women, higher levels of EMs, specifically conjugated estrone and 2- and 4-pathway catechols, were also associated with higher acini count/TDLU. Our data suggest that higher levels of serum EMs are generally associated with lower levels of TDLU involution.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Identification of 16 alpha-hydroxy-estrone as a metabolite of estriol.

During a study on the uptake and retention of estrogens by uterine tissues in postmenopausal women, evidence was obtained of the presence of a metabolite of estriol, tentatively identified as 16 alpha-hydroxy-estrone (16-OHE1). In view of the recent hypothesis concerning the role of 16-OHE1 as a risk marker for breast cancer, attempts were made to establish the identity of the metabolite. After infusions with labelled estriol, radioactive material with chromatographic properties of 16-OHE1 was observed; insufficient material was obtained for micro-recrystallization. After oral administration of estriol, myometrial tissue was extracted, then purified by chromatography and the appropriate fraction was analyzed by gas chromatography-mass spectrometry, monitored at 3 specific mass units. In the women receiving estriol the presence of 16-OHE1 could be unequivocally demonstrated, the concentrations in the myometrium being 6 and 18 ng/g tissue, whereas less than 0.2 ng/g was found in an untreated patient. This identification of 16-OHE1 does not support the hypothesis about its prominent role in human breast cancer. Additional investigations will be necessary to clarify its role in the process of stimulation of estrogen-sensitive tissues under physiological conditions and after exogenous administration of estriol.

Identification of 6 alpha- and 7 alpha-hydroxyestrone as major metabolites of estrone and estradiol in porcine uterus.

Polar metabolites extracted from the effluents of viable porcine uterine strips superfused with either 6,7-3H-estrone or 6,7-3H-estradiol were identified as a 1:1 mixture of 6 alpha-hydroxyestrone and 7 alpha-hydroxyestrone by paper chromatography in various systems, derivatization and crystallizations to a constant specific activity. The hydroxylated compounds are the only derivatives detected after estrone superfusion. The major metabolite of estradiol released in short-time experiments is estrone followed by its 6 alpha- and 7 alpha-hydroxylated derivatives.

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography.

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.

4-hydroxyestrone, isolation and identification in human urine.

Portions of pregnancy and midcycle urines were submitted to hot acid hydrolysis, extracted with benzene/ethyl acetate and the extracts washed with ascorbic acid buffer. From the remaining organic phase the catecholestrogens were removed with borate buffer and further purified on Sephadex LH-20 columns. After derivatisation 4-hydroxyestrone was separated from the isomeric 2-hydroxyestrone peak were identical with that of authentic 4-hydroxyestrone. After treatment of the extracts with sodium borohydride 4-hydroxyestradiol-17 beta was identified by GC-MS. By the addition of trace amounts of tritiated 4-hydroxyestrone a recovery of 40% was calculated. On the basis of this recovery and the peak heights of the gas chromatograms an excretion of 4 microgram (midcycle) and 40 microgram (pregnancy) of 4-hydroxyestrone/24 h was estimated.

A radioimmunoassay method for urinary catechol estrogens.

A radioimmunoassay method for urinary catechol estrogens is described; The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 microgram per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758. 20 microgram per 24 hours.