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CONTEXT: Clinical study of breast cancer patients in Chicago, IL, USA. OBJECTIVE: Ascertain the utility of measurements of single-strand breaks (SSB) in DNA for assessment of breast cancer risk. METHODS: Fine-needle aspirates of the breast, SSB by nick translation, percent breast density (PBD), Gail model risk, cumulative methylation index (CMI), enzymes of DNA repair and tissue antioxidants. RESULTS: DNA repair enzymes and 4-hydroxyestradiol were negatively associated with SSB; CMI and PBD were positively associated. CONCLUSIONS: Quantitative measurement of SSBs by this procedure indicates the relative number of SSBs and is related to promoter methylation, antioxidant availability and percent breast density.

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A high performance liquid chromatography-electrochemical (HPLC-EC) detection method was used to characterize estradiol-17beta (E2) and its metabolites (2-hydroxyE2, 4-hydroxyE2, and 2-methoxyE2) and investigate their seasonal and periovulatory changes in the ovary of the catfish Heteropneustes fossilis. The retention times in minutes of standards determined by individual and mixture applications are: 2-OHE2-6.6, 4-OHE2-7.0, 4-OHE1-11.2, E2-12.0, and 2-methoxyE2-15.2. Since the retention times of 2-OHE2 and 4-OHE2 merged at higher concentrations, the elution peaks of the sample were taken as due to both (2/4-OHE2) for analysis. The steroids were not detectable in the resting and postspawning phases and 2-methoxyE2 was not detectable in the recrudescent (preparatory, prespawning, and spawning) phases as well. E2 and 2/4-OHE2 have maintained an inverse relationship in the recrudescent phase. The E2 concentration was the highest in the preparatory phase (April) with active vitellogenic activity and declined significantly across prespawning and spawning phases (P<0.001, one way ANOVA; P<0.05, Newman-Keuls' test). On the other hand, the concentration of 2/4-OHE2, which was the lowest in the preparatory phase, increased significantly to the peak level in the spawning phase. A single intraperitoneal injection of hCG (100 IU/fish) stimulated significantly the formation of 2/4-OHE2 at 8 h with a simultaneous reduction in E2. 2-MethoxyE2 was detected only after 16 h of the hCG injection. The functional significance of catecholestrogens in the seasonal reproductive cycle and during the hCG-induced ovulation of the catfish was discussed.

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The major detoxification pathway of the carcinogenic catechol estrogens is methylation by catechol- -methyltransferase (COMT). It has been hypothesized that the enzyme encoded by the low-activity allele (COMT(L) ) has a lower catalytic activity for catechol estrogen methylation than that encoded by the high activity allele (COMT(H) ). We expressed and purified human soluble (S)-COMT(H) and S-COMT(L) in and characterized the methylation of 2- and 4-hydroxyestradiol (2- and 4-OH-E2). There were no differences between the kinetic parameters for COMT(H) and COMT(L). The kinetic parameters for S-adenosylmethionine (SAM), the methyl donor in these reactions, also did not differ for COMT(H) and COMT(L). S-adenosylhomocysteine, the demethylated SAM metabolite, inhibited methylation of the catechol estrogens in a non-competitive manner similarly for COMT(H) and COMT(L). Each COMT substrate tested inhibited the methylation of other substrates in a mixed competitive and non-competitive fashion similarly for COMT(H) and COMT(L). Furthermore, in cytosolic fractions of COMT(HH)(MCF-10A and ZR-75-1) and COMT(LL)(MCF-7 and T47D) human breast epithelial cell lines, no differences were detected between the kinetic parameters of COMT with respect to 2- and 4-OH-E2 methylation; nor were COMT protein levels associated with the COMT genotype. These data suggest that the decreased COMT enzymatic activity that has been detected in human tissue in association with the COMT(L) allele is not reflected by differences in the affinity or capacity of COMT(H) and COMT(L) for catechol estrogen methylation. These results raise the question of what accounts for the difference in COMT activity associated with the COMT(HH) and COMT(LL) genotypes in human tissue.

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Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances. However, some compounds do not show enough sensitivity in LC/MS and soft-ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information. This report presents an overview of the derivatization methods in the LC/MS analysis of neurosteroids or neuroactive neurosteroids, which are synthesized and accumulated in the nervous system. The derivatization of pregnenolone 3-sulfate, one of these steroids, with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole gave a satisfactory sensitivity during the quantitative analysis using LC/ESI-MS. The obtained results are much lower than those previously obtained using gas chromatography/mass spectrometry or radioimmunoassay. On the other hand, the derivatization to acetate was useful for the treatment of labile catechol estrogens in rat brains and gave enough structural information in LC/APCI-MS, which confirmed the existence of catechol estrogens in mammalian brains.

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A method for the analysis of N-acetylcysteine conjugates of catechol estrogens [catechol estrogen mercapturates (CE SRs)], which are likely to be urinary markers of estrogen-induced tumors, was established in this study. The characteristics of the method that was established were (1) cleanup of urine using the immunoaffinity column of CE SRs, (2) detection of catechol estrogens (CEs) and CE SRs by electrochemical detection, which provided the high specificity, and (3) stability of CE SRs through the cleanup. Using this method, the simultaneous quantitation of 2-hydroxy-17beta-estradiol (2-OHE(2)), 4-hydroxy-17beta-estradiol (4-OHE(2)), 2-hydroxyestrone (2-OHE(1)), 4-hydroxyestrone (4-OHE(1)), 2-hydroxyestrone 1-N-acetylcysteine thioether (2-OHE(1) 1SR), 2-hydroxyestrone 4-N-acetylcysteine thioether (2-OHE(1) 4SR), and 4-hydroxyestrone 2-N-acetylcysteine thioether (4-OHE(1) 2SR) in the range of 1-15 ng was performed. We first demonstrated the presence of CE SRs, 2-OHE(1) 1SR and 2-OHE(1) 4SR, in urine from rats treated intraperitoneally with 17beta-estradiol (E(2)) at a dose of 5 mg/kg. In female rats, the amount of 2-OHE(1) 1SR was several-fold greater than that of 2-OHE(1) 4SR, while the presence of 4-OHE(1) 2SR was not confirmed. The level of CEs and CE SRs in male rats was approximately (1)/(2)-(1)/(20) of that in female rats. The excretion rate following administration of 2-OHE(1) at 2 mg/kg and that following the administration of 4-OHE(1) at 2 mg/kg were different in female rats. In addition, 4-OHE(1) 2SR was present in the urine of male Syrian hamsters treated intraperitoneally with E(2), whereas it was absent in rats.

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The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.

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Collisionally activated decompositions (CAD) of [M + H]+ ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b2, modified y2, y2, and b1 ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C-S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.

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The catechol estrogens (CE), 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) were analyzed for their binding affinity to the estrogen receptor of MCF-7 cells. Applying a competitive binding assay to cytosols prepared from MCF-7 breast cancer cells, we measured a relative binding affinity of 23% (2-OH-E2) and 26% (4-OH-E2) compared to E2. Nuclear binding assays with the same cell line demonstrated a high specific binding with Kd's of 0.31 nM (2-OH-E2) and 0.21 nM (4-OH-E2). The relative binding affinity measured was 25% and 42% for 2-OH-E2 and 4-OH-E2, respectively. Based on this nuclear binding it can be concluded that the estrogen receptor occupied by CE is bound within the nucleus and might therefore be transcriptionally active.

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Catecholestrogens (CCEs), namely 2- or 4-hydroxyestradiol and hydroxyestrone, are highly polar, reactive, and extremely labile estrogen metabolites in many experimental conditions. For these reasons, indirect assay methods mainly have been used. Some experimental evidence suggests that CCEs are synthesized and biologically active mostly in target cells. At this level, unfortunately, the indirect assays cannot be used. We present a method of gas chromatographic/mass spectral (GC/MS) analysis for the identification of individual CCEs; the major fragmentation ions of authentic estrogen standards as trimethylsilylether derivatives, and the MS patterns of the major CCEs, namely, 2-hydroxyestradiol and hydroxyestrone, are included. Few examples of CCEs detected in human breast cancer tissues and in breast cyst fluids are reported. Sample extracts were submitted to reversed-phase, high-performance liquid chromatography (RP-HPLC) and were quantified by "on line" electrochemical (EC) detection; thereafter, either crude extracts or single eluted peaks were submitted to GC/MS, by which detection limits of less than 5 pmol were attained. As expected, the molecular ion was the most relevant molecule in all but one case. On the contrary, the other relative intensities of major fragmentation ions M -15, M -30, M -90, and M -15 + (-90) were unevenly distributed, although represented in the majority of cases. In all cases, the GC/MS of peak fractions, purified by RP-HPLC and UV detection, confirmed the results of liquid chromatographic analysis combined with EC detection. In contrast, GC/MS of crude extracts was not equally satisfactory. Comparison of a liquid chromatography system with EC detection and the GC/MS approach revealed some inconsistency in quantitation of individual CCEs.(ABSTRACT TRUNCATED AT 250 WORDS)

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To assess the role of catechol estrogens in the initiation of labor, we compared the levels in amniotic fluid during the second and third trimesters and from women undergoing cesarean section at term not in labor and those with spontaneous labor at term. Catechol estrogen concentrations in amniotic fluid increased significantly with the progress of pregnancy. Further, concentrations (mean +/- SE) were significantly higher in spontaneous labor at term (468.6 +/- 29.5 pg/ml) compared with those obtained during cesarean section (242.6 +/- 22.3 pg/ml) at term not in labor. We suggest that catechol estrogens, through their stimulating effects on prostaglandin synthesis, participate in the initiation of labor.

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The synthesis and use of stable isotope-labeled analogs of various steroids have made it possible to undertake a study of follicular fluid (FF) aspirated from mature and preovulatory follicles. Our previous results have been brought together here in order to review quantitative work done by gas chromatography-mass spectrometry. A positive gradient between peripheral plasma and FF concentrations of a steroid suggests the possibility of ovarian biosynthesis. This is particularly relevant to the catecholestrogens, 19-norsteroids, and some corticosteroids.

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The application of inclusion chromatography with beta-cyclodextrin as a mobile phase additive in high-performance liquid chromatography to the determination of in vitro metabolites of estriol is reported. Compared to conventional methods, the inclusion chromatography gives much more satisfactory separation of isomeric estriol derivatives in a short time. Species difference (rat and guinea pig liver homogenates) is observed in the glucuronidation of estriol. The hydroxylation of estriol with rat liver homogenate occurs preferentially at the 2-position. Methylation of this product with catechol-O-methyl transferase in rat liver gives almost equal amounts of 2- and 3-methyl ethers. In contrast, 4-hydroxyestriol gives 4-methyl ether as a main product.

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4-Hydroxyestradiol bearing a 3H label specifically at C-2 was prepared chemically and incubated with male rat liver microsomes or mushroom tyrosinase. A very high proportion (80-90%) of the 3H was displaced from the labeled steroid when either glutathione or N-acetylcysteine was present, and tyrosinase was shown not to require NADPH as cofactor for this reaction. In either case, only negligible amounts (less than 3%) of the 3H radioactivity were found associated with water-soluble adducts in contrast to 3H-labeled 2-hydroxyestradiol, which gave rise to about 25% of such products. The effect of ascorbic acid on the microsomal reaction with regiospecifically labeled estradiol, 2-hydroxyestradiol, and 4-hydroxyestradiol was also investigated, and the results are discussed in terms of the reactivity at different carbon atoms in ring A of the catechol estrogens. All the evidence points to conjugation of 4-hydroxyestradiol with glutathione or N-acetylcysteine at C-2 but not C-1 of this highly reactive catechol estrogen. Measuring the displacement of 3H as 3H2O from specific positions in the steroid ring provides a useful and sensitive method to assess the formation of adducts in cases where their isolation and characterization is particularly difficult.

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Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) and catecholestrogen were localized in the parotid gland of the rat by immunocytochemical methods. Specific immunoreactive deposits for COMT and catecholestrogen were found in the cytoplasm of duct cells, but only those for COMT in myo-epithelial cells. The pattern of localization of COMT and catecholestrogen in the parotid gland suggests a functional relationship between COMT and catecholestrogen.

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This paper presents a short review of the results obtained to date in our laboratory, with respect to the studies of steroid excretion profiles in both breast and endometrial cancer patients, by using gas-liquid chromatographic analysis. These data demonstrate the importance of minor estrogens, including catechol estrogens, and of their ratios with the classical ones, in studies of steroid metabolism in both breast and endometrial cancer. New data concerning postmenopausal endometrial cancer are consistent with our previous observations and demonstrate the necessity of measuring these steroids directly in tumors and examining patterns of metabolism in vitro. In order to analyze steroid metabolic patterns in vitro, however, high-performance liquid chromatography rather than gas-liquid chromatography methods are preferable on account of their selectivity, specificity, sensitivity and capacity to handle labile materials. With the aim of providing methods suitable for the complete resolution and analysis of these complex natural mixtures a method of computer-aided optimization of HPLC has been developed and its practical utility has been tested.

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In the present study, we have confirmed the existence of a biphasic response in striatal dopamine receptor sensitivity following the administration of estradiol benzoate (EB). This biphasic response consists of a hyposensitive phase 24 h after the last injection of EB, followed by a hypersensitive phase 72 h after the last injection of EB. In contrast to this, the administration of 2-hydroxyestradiol (2-OHE2), a catechol metabolite of estrogen, resulted in a striatal dopamine receptor hypersensitivity at both 24 and 72 h after the last injection of 2-OHE2. Studies on the in vivo metabolism of [3H]estradiol to its [3H]catechol metabolites indicated that the administration of piperonyl butoxide (PBO; a microsomal enzyme inhibitor) significantly decreased the level of [3H]catechol metabolites of [3H]estradiol in the striatum and in the medial basal hypothalamus. In addition, PBO administration resulted in about a 7-fold decrease in the ability of estradiol to induce a striatal dopamine receptor hypersensitivity. These data indicate that the biphasic response in striatal dopamine receptor sensitivity following estrogen, may be mediated by separate molecular mechanisms. The association of the hypersensitive phase with pharmacological doses and/or treatment paradigms, the development of a similar hypersensitivity following the administration of the 2-OHE2 metabolite of estrogen and the attenuation of the estrogen-induced striatal dopamine receptor hypersensitivity in PBO pretreated animals all suggest that this striatal dopamine receptor hypersensitivity may be mediated, at least in part, by the catecholestrogens.

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2-Hydroxyethynyloestradiol (2-OHEE2), a major biliary metabolite of 17 alpha-ethynyloestradiol in female rats, is conjugated largely with glucuronic acid. Accurate quantitation of [3H]2-OHEE2 deconjugated by enzymic hydrolysis depends upon co-incubation with ascorbate (5-10 mM). In the absence of ascorbate, the proportion of [3H]2-OHEE2 declines by 30 +/- 7% (x +/- SD, n = 4) during a 3 h incubation of bile with arylsulphohydrolase and beta-glucuronidase. Over 16 h, decomposition of the catechol leads to a decrease in ether-extractable 3H labelled components.

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