Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1.

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3ß, 17ß-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low µM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17ß-dihydroequilenin (17ß-Eqn), 17ß-dihydroequilin (17ß-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17ß-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17ß-Eq was stimulated at celecoxib concentrations below 40 µM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

[Fragmentation pathways of five estrogens using electrospray ionization quadrupole time-of-flight mass spectrometry].

The fragmentation pathways of five estrogens (estradiol, estrone, equilin sulfate, 17 a-dihydroequilin sulfate and equilenin sulfate) have been studied with high resolution and high mass accuracy using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS) in the negative ion mode. Molecular weights were obtained from [M-H](-) ions in the product ion spectra. The results indicate that the five structurally similar estrogens have similar fragmentation pathways. Using their stable isotope forms as internal reference compounds, the accurate mass and composition of the fragment ions were determined. During collision-induced dissociation (CID), cleavage is initiated by loss of oxygen atoms from carbon-17, after which D and C rings cleave sequentially and rearrange to finally form stable conjugate structures with highly abundant characteristic fragment ions at m/z 183 (accompanied by m/z 181), m/z 169 and m/z 145 (accompanied by m/z 143). Understanding these characteristic fragmentation pathways of estrogens will be helpful in identifying the structures of steroid hormones in general.

Differentiating isobaric steroid hormone metabolites using multi-stage tandem mass spectrometry.

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17ß-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus ß-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

Ozonation of a mixture of estrogens and progestins in aqueous solution: interpretation of experimental results by computational methods.

The degradation of the mixture of steroid hormones including seven estrogens (17α-estradiol, 17ß-estradiol, 17α-dihydroequilin, 17α-ethinyl estradiol, estriol, estrone and equilin) and five progestins (levonorgestrel, gestodene, trimegestrone, medrogestone and progesterone) by ozonation in aqueous solution is investigated. The ozonation process provides high removal (up to 100%) of hormones and estrogenicity in the treated water. Computational methods such as quantum chemistry calculations (QCCs) are applied to interpret the observed results. Quantum chemistry descriptors computed for steroid hormones explain the nature of the reactions and differences in reactivities between estrogen and progestin hormones within the framework of the Density Functional Theory (DFT). Computed molecular descriptors were combined with physical properties to develop qualitative structure activity relationship (QSAR) models (using multiple linear regression algorithm). The developed models have correlation coefficients (R(2)) of 0.994 for estrogens and 0.997 for progestins, and could be used to predict the removal efficiencies for similar compounds. The frontier molecular orbitals (the HOMO and the LUMO) have a major impact on the reactivity of steroid hormones. The susceptibility of certain functional groups to ozone and possible reactive sites for all steroids was discussed by Frontier Molecular Orbital approach.

Occurrence of estrogen hormones in biosolids, animal manure and mushroom compost.

The presence of natural estrogen hormones as trace concentrations in the environment has been reported by many researchers and is of growing concern due to its possible adverse effects on the ecosystem. In this study, municipal biosolids, poultry manure (PM) and cow manure (CM), and spent mushroom compost (SMC) were analyzed for the presence of seven estrogen hormones. 17α-estradiol, 17ß-estradiol, 17α-dihydroequilin, and estrone were detected in the sampled biosolids and manures at concentrations ranging from 6 to 462 ng/g of dry solids. 17α-estradiol, 17ß-estradiol, and estrone were also detected in SMC at concentrations ranging from 4 to 28 ng/g of dry solids. Desorption experiments were simulated in the laboratory using deionized water (milli-Q), and the aqueous phase was examined for the presence of estrogen hormones to determine their desorption potential. Very low desorption of 0.4% and 0.2% estrogen hormones was observed from municipal biosolids and SMC, respectively. An estimate of total estrogen contribution from different solid waste sources is reported. Animal manures (PM and CM) contribute to a significant load of estrogen hormones in the natural environment.

Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

Influence of alkalinity and salinity on the sonochemical degradation of estrogen hormones in aqueous solution.

Ultrasound assisted degradation of estrogen hormones was examined in a batch reactor using a 2 kW (20 kHz) sonication unit. The degradation of estrogens follow a pseudo first order rate kinetics, and the order of degradation is 17alpha-dihydroequilin > equilin >17alpha-ethinyl estradiol >17alpha-estradiol >17beta-estradiol > estrone > estriol. Effect of solution alkalinity and salinity on the sonochemical degradation of estrogen hormones is examined. At alkalinity concentration of 10 mM, no adverse effect on the degradation rate constants of estradiols (17alpha-estradiol, 17beta-estradiol, and 17alpha-ethinyl estradiol) was observed, whereas equilin compounds showed a decrease in their degradation rate constants. Significant inhibitory effects were observed for all the compounds at high alkalinity concentration of 120 mM and which could be due to the scavenging of OH(*) radicals in the bulk solution. The presence of salinity (0.17 M) enhanced the estrogen degradation except for the equilin compounds. Simultaneous presence of high alkalinity (120 mM) and salinity (0.17 M) also increased the degradation of estrogen hormones than the case when only alkalinity (120 mM) was present, indicating the diffusion of analytes to the cavity interface where most of the degradation occurs under these conditions. A mechanistic approach was used to model the degradation behavior of estrogen hormones under different solution alkalinity and salinity conditions.

Molecular clustering of endometrial carcinoma based on estrogen-induced gene expression.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Free synthetic and natural estrogen hormones in influent and effluent of three municipal wastewater treatment plants.

Three municipal wastewater treatment plants (WWTPs) in southeastern Pennsylvania were sampled to determine the presence and concentrations of 12 natural and synthetic estrogen hormones in the wastewater influent and effluent. The target estrogens were 17alpha-estradiol, estrone, estriol, equilin, 17alpha-dihydroequilin, 17beta-estradiol, 17alpha-ethinyl estradiol, gestodene, norgestrel, levonorgestrel, medrogestone, and trimegestone. One WWTP uses a biofilm reactor (packed-bed trickling filter),and the other two use suspended-growth media (continuously stirred activated sludge reactor and sequential batch reactor). Estrone was detected in all the three plants; estriol and estradiol were detected at two WWTPs; and 17 alpha-dihydroequilin and 17 alpha-ethinyl estradiol were detected at one WWTP. The concentration of estrogens in the influent and effluent of the three treatment plants ranged from 1.2 to 259 ng/L and 0.5 to 49 ng/L, respectively. The percentage removal of estrogens from the aqueous phase ranged from 41 to 99%, except in the case of 17alpha-dihydroequilin; the removal of 17alpha-dihydroequilin was negligible. The suspended-growth media systems showed higher removal efficiencies for estrogens than the biofilm system. The analytical method uses a Varian C-18 solid-phase extraction (Varian Inc., Palo Alto, California), followed by a derivatization with bis(trimethylsilyl)trifluoroacetamide. The detection limits for the estrogen compounds ranged from 0.1 to 10 ng/L using a sample size of 1 L. The method recoveries ranged from 71 to 120%, and the relative standard deviation ranged from 6 to 14% for all the hormones.

The estrogen metabolite 17beta-dihydroequilenin counteracts interleukin-1alpha induced expression of inflammatory mediators in human endothelial cells in vitro via NF-kappaB pathway.

In most studies showing cardio- and vasculoprotective effects of estrogens, 17beta-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17alpha-dihydroequilenin, 17beta-dihydroequilenin, delta-8,9-dehydroestrone, estrone and 17beta-estradiol and stimulated with interleukin (IL)-1alpha. The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17beta-dihydroequilenin and 17beta-estradiol at a concentration of 1 microM reduced IL-1alpha-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17alpha-dihydroequilenin and delta-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10 microM whereas estrone had no effect. The effect of 17beta-dihydroequilenin on IL-1alpha-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17beta-dihydroequilenin also inhibited IL-1alpha-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17beta-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens--as shown in our study--could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases.

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.

Mixtures of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents.

Most effluents from wastewater treatment works (WwTWs) contain estrogenic chemicals that include steroidal estrogens and xenoestrogens. We investigated the nature of mixtures of estrogenic contaminants taken up by two species of fish exposed to two WwTWs effluents. Sexually immature rainbow trout, Oncorhynchus mykiss, and sexually mature roach, Rutilus rutilus, were exposed to tap water, river water, or one of two estrogenic WwTWs effluents for up to 10 days, when the fish were sacrificed and tissues removed for chemical analysis. Estrogenic contaminants in the bile and gonads were hydrolyzed, concentrated by solid-phase extraction, and fractionated by RP-HPLC. Active fractions were detected and quantified using a yeast estrogen receptor transcription screen (YES assay) and the identities of estrogenic components in the fractions determined by GC-MS. Bile from rainbow trout exposed to either tap water or river water contained low amounts of 17beta-estradiol (E2) and estrone (E1) with a total estrogenic activity (mean+/-standard error) of 10+/-5 and 31+/-9 ng of E2 equivalents/mL (ng of E2eq/mL) for male and female fish, respectively. In effluent-exposed trout the total estrogen content of bile was considerably higher with the following composition and concentrations (ng of E2eq/mL) of individual estrogens: E2 (male, 591+/-125; female, 710+/-207), E1 (male, 338+/-75; female, 469+/-164), ethinylestradiol, EE2 (male, 32+/-2; female, 40+/-6), nonylphenol (NP) and short-chain NP polyethoxylates (male, 21+/-4; female, 22+/-3). An additional estrogenic compound, 17beta-dihydroequilenin (DHQ), was identified for the first time in effluent-exposed fish, and was present in trout bile at concentrations of (male) 40+/-9 and (female) 30+/-5 ng of E2 eq/mL. DHQ, E2, E1, and EE2, but not NP or NP polyethoxylates, were also detected in bile of effluent-exposed roach, and the concentrations of all these steroidal estrogens in ng of E2eq/mL were lower in male (E2, 62+/-2; E1, 35+/-11; EE2, 10+/-2; DHQ, 1+/-1) compared with female (E2, 740+/-197; E1, 197+/-37; EE2, 40+/-6; DHQ, 8+/-2) roach. The synthetic estrogen EE2 was also detected in the testes and ovaries of effluent-exposed roach. This study shows that a mixture of estrogenic contaminants present in WwTWs effluents bioconcentrate in fish tissues, resulting in the induction of vitellogenin, and are likely to contribute to feminizing effects in wild fish living in U.K. rivers. The composition of the mixture of estrogenic contaminants in the bile is species dependent and may determine the susceptibility of fish to the effects of exposure to estrogenic effluents.

Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa.

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.

B-ring unsaturated estrogens: biological evaluation of 17alpha-Dihydroequilein and novel B-Nor-6-thiaequilenins as tissue selective estrogens.

The pharmacology and SAR of representative equine estogens is described. 17alpha-Dihydroequilenin was found to prevent bone loss after 5 weeks of oral administration to ovariectomized rats. The stereochemical significance of the D-ring and the C/D ring juncture was investigated with a series of benzothiophene-based equilenin analogues.

Comparison of the proliferative effects of estradiol and conjugated equine estrogens on human breast cancer cells and impact of continuous combined progestogen addition.

OBJECTIVES: So far, most epidemiological studies investigating breast cancer risk and hormone replacement therapy have been conducted with conjugated equine estrogens (CEE). Recent trials indicate that the addition of progestogens may increase breast cancer risk. In the present study, we compared the effects of the human estrogen 17beta-estradiol (E(2)) with those of the main equine components of CEE, i.e. equilin (Eq) and 17alpha-dihydroequilin (Dheq) on the proliferation of human breast cancer cells. The proliferative effect of progestogen addition was also investigated. MATERIALS AND METHODS: The well-established human breast cancer cell line MCF-7 was used as an in vitro model. The proliferative effect of E(2), Eq and Dheq was tested in the concentration range 0.01-10 nmol/l. The progestogens progesterone, medroxyprogesterone acetate (MPA) and norethisterone (NET) were continuously combined with 0.1 nmol/l estrogen at concentrations of 0.01 nmol/l, 1 nmol/l, 0.1 mumol/l and 10 mumol/l. Proliferation was measured after 7 days by the adenosine triphosphate (ATP) chemosensitivity test. RESULTS: All three estrogens increased the proliferation of MCF-7 cells by between 40 and 180%. The most proliferatively potent estrogen was E(2), followed by Eq and Dheq, which showed a slightly lower proliferative activity than E(2). The addition of progesterone inhibited E(2)-induced proliferation by about 30%, but only at the high non-physiological concentration of 10 mumol/l. All three progestogens inhibited Eq-induced proliferation, although their effect tended to be low, with values between 5 and 40%. No progestogen reduced Dheq-induced proliferation by more than 20%. In contrast, MPA slightly increased the proliferation rate by about 5% at the high physiological concentration of 0.1 mumol/l when combined with Dheq. The same held true when MPA and NET were added at the high pharmacological concentration of 10 mumol/l, causing increases of about 10%. CONCLUSIONS: Our results indicate that equine estrogens have a proliferative action similar to that of 17beta-estradiol. Continuous addition of progestogens does not result in any major reduction of proliferative potency. Some progestogens may even enhance the estrogen-induced proliferation of pre-existing breast cancer cells, particularly when combined with certain equine estrogens. However, in none of the tested circumstances do progestogens increase the proliferative effect of estradiol, and progesterone has no deleterious effect even at pharmacological levels, in contrast to progestogens.

Equine estrogens differentially prevent neuronal cell death induced by glutamate.

OBJECTIVE: In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta(8)-estrone(,) and Delta(8),17beta-estradiol against glutamate toxicity. METHODS: The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay. RESULTS: The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutamate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroequilenin > 17alpha-dihydroequilenin > equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > equilenin = 17beta-dihydroequilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroequilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells. CONCLUSIONS: Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.

Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells.

Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT). Equilin and equilenin are the major components of the widely prescribed drug used for ERT. These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages. To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF, was exposed to 4-OHEQ and transfected into human fibroblast. Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing. The proportion of plasmids with the mutated supF gene was increased dose-dependently. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions. Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions. The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site. C:G --> G:C and C:G --> A:T mutations were detected preferentially with lesser numbers of C:G --> T:A transitions. Sixty-two percent of base substitutions were observed particularly at C:G pairs in (5')-TC/AG-(5') sequences. Using (32)P-post-labeling/gel electrophoresis analysis, 4-OHEN-dC was a major adduct, followed by lesser amounts of 4-OHEN-dA adduct. Mutations observed at C:G pairs may result from 4-OHEN-dC adduct. These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in (5')-TC/AG-(5') sequences.

Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium.

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

Pharmacokinetics of 17 beta-dihydroequilin sulfate in normal postmenopausal women under steady state conditions.

OBJECTIVE: In the present study, the constant infusion of [3H]17 beta-dihydroequilin sulfate ([3H]17 beta-EqS) was used to estimate the metabolic clearance rate (MCR) of 17 beta-dihydroequilin sulfate (17 beta-EqS) and to measure the conversion of this estrogen to equilin sulfate (EqS), equilenin sulfate (EqnS), 17 beta-dihydroequilenin sulfate (17 beta-EqnS), equilin (Eq), equilenin (Eqn), 17 beta-dihydroequilin (17 beta-Eq), and 17 beta-dihydroequilenin (17 beta-Eqn) in normal postmenopausal women. METHODS: In seven healthy postmenopausal women, infusion of [3H]17 beta-EqS was started 30 minutes after a priming dose and continued at a constant rate of 10-20 microCi/hour, for 3-6 hours. Three blood samples were taken during and at the end of infusion. From the plasma, unconjugated and sulfate-conjugated estrogens, 17 beta-EqS, EqS, EqnS, 17 beta-EqnS, Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated and purified by high performance liquid chromatography. The MCR of 17 beta-EqS and the conversion ratios and transfer constants (rho) for precursor (17 beta-EqS) to products were calculated. RESULTS: The mean MCR of 17 beta-EqS was calculated to be 797 +/- 90 L/day or 506 +/- 60 L/m2 per day. The mean conversion ratio (CRPRE-PROBB) was 2.4 +/- 0.4 for EqS, 0.3 +/- 0.04 for EqnS, 0.25 +/- 0.03 for 17 beta-EqnS, 0.09 +/- 0.02 for Eq, 0.03 +/- 0.01 for Eqn, 0.08 +/- 0.02 for 17 beta-Eq, and 0.03 +/- 0.01 for 17 beta-Eqn. In both the sulfate-conjugated and unconjugated forms, the most abundant metabolite formed was Eq. Based on the previously reported MCR of EqS (170 L/m2 per day) and 17 beta-Eq (1252 L/m2 per day), the transfer constants [rho]BB were calculated to be 0.8 +/- 0.10 and 0.20 +/- 0.03, respectively. The results indicate that a large portion of 17 beta-EqS is converted to EqS and the more potent estrogen 17 beta-Eq. The ratio of rho EqS-17 beta-EqS to rho 17 beta-EqS-EqS was calculated to be 0.8 +/- 0.1 and represents the extent of C-17-oxidation and reduction and indicates that substantial amounts of 17 beta-reduced metabolites will still be present in the blood although the oxidation reaction was somewhat greater. CONCLUSION: The data indicate that, compared with the classic estrogens, the in vivo metabolism of ring B unsaturated estrogens is complex. Thus, although the amount of 17 beta-EqS originally present in the conjugated equine estrogens is small, the pharmacokinetics and pharmacodynamics of EqS, 17 beta-EqS, and the extensive interconversions between these estrogens support the hypothesis that the major in vivo activity of the EqS present in conjugated equine estrogens is expressed through its metabolism to 17 beta-EqS and 17 beta-Eq. Furthermore, the increased estrogenic activity associated with this drug may in part be due to the formation of these 17 beta-reduced metabolites.

Equine estrogens induce apolipoprotein E and glial fibrillary acidic protein in mixed glial cultures.

Premarin, which contains several equine estrogens, as well as estradiol (E2) as a minor component, is widely used for replacement therapy of estrogen deficits, but little is known of its direct actions on brain cells. In mixed glial cultures, apolipoprotein E (apoE) and glial fibrillary acidic protein (GFAP) are induced by estrogens. GFAP induction showed an inverted-U shape E2 dose response, with a maximum induction at 1 pM, whereas apoE mRNA induction was greatest at 100 pM. GFAP and ApoE mRNAs were induced by equine estrogens in the following order: E2=equilin>estrone>17 alpha-dihydroequilenin. However, the induction of apoE secretion by 17 alpha-dihydroequilenin was as effective as by the other estrogens. The greater response of apoE secretion than GFAP mRNA induction to 17 alpha-dihydroequilenin might be therapeutically important because of the glial scarring during brain lesions, in which GFAP induction has a major role in inhibiting neurite outgrowth, whereas apoE secretion supports neurite outgrowth.