Developmental toxicity and transcriptome analysis of equine estrogens in developing medaka (Oryzias latipes) using nanosecond pulsed electric field incorporation.

Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and 3855 total differentially expressed genes (DEGs), 1117 DEGs overlapped between Eq. (55.4 % of total DEGs) and Eq. (29.0 % of total DEGs). Gene ontology indicated effects in terms related to blood circulation and cell junctions. Pathway analyses using DEGs revealed that both Eq and Eqn treatments at 10 mg/L affected various KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, mitogen-activated protein kinase signaling, retinol metabolism, and cytokine-cytokine receptor interaction. These results suggest that the disruption of these KEGG pathways is involved in the developmental toxicity of EQs in medaka embryos.

PGRMC1 can trigger estrogen-dependent proliferation of breast cancer cells: estradiol vs. equilin vs. ethinylestradiol.

Objective: Previous studies have shown that progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer tissue can predict a worse prognosis for breast cancer patients. Moreover, we demonstrated that PGRMC1 can increase the proliferation of progestogens. However, the role of PGRMC1 in terms of estrogen-induced proliferation and comparing different estrogens is still unclear. Methods: Non-transfected and PGRMC1-transfected T-47D cells were stimulated with estradiol (E2), with equilin (EQ), or with ethinylestradiol (EE) at 1, 10, and 100 nmol/l. Increase of proliferation was compared with a control (without estrogens) and with the estrogen-induced stimulation in empty vector cells vs. PGRMC1-transfected cells. Results: The empty vector cells showed significant proliferation (12-15%) with all three estrogens only at the highest concentration, with no relevant differences between the estrogens. PGRMC1-transfected cells showed about three-fold higher proliferation (29-66%), whereby E2 elicited the strongest and EE the lowest proliferating effects, significantly lower compared to E2 and also compared to EQ. No significant differences were seen between E2 and EQ. Conclusions: PGRMC1 increases strongly the estrogen-dependent breast cell proliferation. The proliferating effects of EE may be lower compared to E2 and EQ. This could have importance in comparing hormone therapy and contraception. Thus, PGRMC1 not only could predict the risk using progestogens but also of different estrogens.

Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling.

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17ß-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)ß expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERß or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

Equilin displays similar endothelium-independent vasodilator potential to 17ß-estradiol regardless of lower potential to inhibit calcium entry.

Conjugated equine estrogens (CEE) have been widely used by women who seek to relieve symptoms of menopause. Despite evidence describing protective effects against risk factors for cardiovascular diseases by naturally occurring estrogens, little is known about the vascular effects of equilin, one of the main components of CEE and not physiologically present in women. In this regard, the present study aims to compare the vascular effects of equilin in an experimental model of hypertension with those induced by 17ß-estradiol. Resistance mesenteric arteries from female spontaneously hypertensive rats (SHR) were used for recording isometric tension in a small vessel myograph. As effectively as 17ß-estradiol, equilin evoked a concentration-dependent relaxation in mesenteric arteries from female SHRs contracted with KCl, U46619, PDBu or ET-1. Equilin-induced vasodilation does not involve classical estrogen receptor activation, since the estrogen receptor antagonist (ICI 182,780) failed to inhibit relaxation in U46619-precontracted mesenteric arteries. Vasorelaxation was not affected by either endothelium removal or by inhibiting the release or action of endothelium-derived factors. Incubation with L-NAME (NOS inhibitor), ODQ (guanylyl cyclase inhibitor) or KT5823 (inhibitor of protein kinase G) did not affect equilin-induced relaxation. Similarly, indomethacin (COX inhibitor) or blockage of potassium channels with tetraethylammonium, glibenclamide, 4-aminopyridine, or ouabain did not affect equilin-induced relaxation. Inhibitors of adenylyl cyclase SQ22536 or protein kinase A (KT5720) also had no effects on equilin-induced relaxation. While 17ß-estradiol inhibited calcium (Ca2+) -induced contractions in high-K+ depolarization medium in a concentration-dependent manner, equilin induced a slight rightward-shift in the contractile responses to Ca2+. Comparable pattern of responses were observed in the concentration-response curves to (S)-(-)-Bay K 8644, a L-type Ca2+ channel activator. Equilin was unable to block the transitory contraction produced by caffeine-induced Ca2+ release from intracellular stores. In conclusion, equilin blocks L-type Ca2+ channels less effectively than 17ß-estradiol. Despite its lower effectiveness, equilin equally relaxes resistance mesenteric arteries by blocking Ca2+ entry on smooth muscle.

Occurrence and seasonal variation of equine estrogens, equilin and equilenin, in the river water of Japan: Implication with endocrine-disrupting potentials to Japanese medaka (Oryzias latipes).

In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC-MS/MS analysis revealed that Eq concentrations were 2.7 ±â€¯6.7, 0.22 ±â€¯0.12, and 1.2 ±â€¯0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka.

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.

Endocrine-disrupting potentials of equine estrogens equilin, equilenin, and their metabolites, in the medaka Oryzias latipes: in silico and DNA microarray studies.

Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17ß-dihydroequilin, 17α-dihydroequilenin and 17ß-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17ß-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17ß-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.

[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.

Fragmentation pathways of five estrogens using electrospray ionization quadrupole time-of-flight mass spectrometry / 药学学报

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.

Modelling defined mixtures of environmental oestrogens found in domestic animal and sewage treatment effluents using an in vitro oestrogen-mediated transcriptional activation assay (T47D-KBluc).

There is growing concern of exposure of fish, wildlife and humans to water sources contaminated with oestrogens and the potential impact on reproductive health. Environmental oestrogens can come from various sources including concentrated animal feedlot operations (CAFO), municipal waste, agricultural and industrial effluents. US EPA's drinking water contaminant candidate list 3 (CCL3) includes several oestrogenic compounds. Although these contaminants are currently not subject to any proposed or promulgated national primary drinking water regulations, they are known or anticipated to occur in public water systems and may require future regulation under the Safe Drinking Water Act. Using an in vitro transcriptional activation assay, this study evaluated oestrogens from CCL3 both individually and as a seven oestrogen mixture (fixed ray design) over a broad range of concentrations, including environmentally relevant concentrations. Log EC(50) and Hillslope values for individual oestrogens were as follows: estrone, -11.92, 1.283; estradiol-17α, -9.61, 1.486; estradiol-17ß, 11.77, 1.494; estriol, -11.14, 1.074; ethinyl estradiol-17α, -12.63, 1.562; Mestranol, -11.08, 0.809 and Equilin, -11.48, 0.946. In addition, mixtures that mirrored the primary oestrogens found in swine, poultry and dairy CAFO effluent (fixed-ratio ray design), and a ternary mixture (4 × 4 × 4 factorial design) of oestrogens found in hormone replacement therapy and/or oral contraceptives were tested. Mixtures were evaluated for additivity using both the concentration addition (CA) model and oestrogen equivalence (EEQ) model. For each of the mixture studies, a broad range of concentrations were tested, both above and below environmentally relevant concentrations. Results show that the observed data did not vary consistently from either the CA or EEQ predictions for any mixture. Therefore, either the CA or EEQ model should be useful predictors for modelling oestrogen mixtures.

Application of density functional theory (DFT) to study the properties and degradation of natural estrogen hormones with chemical oxidizers.

Estrone (E1), 17ß-estradiol (E2), estriol (E3), equilin (EQ) and 17α-estradiol (17α) estrogen hormones are released by humans and animals and have been detected in the environment and municipal wastewater treatment plants. The structural and electronic properties of natural hormone molecules are investigated by performing density functional theory calculations and used to predict their properties and chemical behavior. Quantitative structure property relationship (QSPR) approach is applied to correlate the estrogenicity associated with the natural estrogen hormones according to their molecular properties. The obtained relationship reveals the importance of the frontier molecular orbital energy in the interpretation of estrogenic activity of hormones, which is consistent with the previous research. Moreover, the obtained molecular descriptors also aid determination of the degradability of hormones, and to rationalize degradation pathways, with chemical oxidizers such as ozone and hydroxyl radical. Both types of interactions belong to the orbital-controlled reactions. The active sites determined by Fukui functions for the estrogen hormone molecules confirm the reaction pattern that initiates the attack of the aromatic ring for both ozone and hydroxyl radical. The reactive sites of the molecules are mapped with subsequent reaction intermediates and compared with experimental data obtained from the literature.

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.

Cellular and molecular actions displayed by estrone on vascular endothelium.

In this work we provide evidence that estrone "per se" modulates cellular endothelial growth and survival, events that play key roles in the development of vascular disease. Moreover, under oxidative stress conditions the hormone prevented apoptosis triggered by hydrogen peroxide. Although estrone did not affect E-selectin and VCAM-1 mRNAs synthesis, the hormone prevented the expression of these adhesion molecules induced by the proinflammatory agent LPS. The steroid partially attenuated leukocyte adhesion not only under basal conditions but also in the presence of LPS. Using ICI182780 compound as estrogen receptor antagonist, and PD98059 as MAPK inhibitor we obtained evidence that the mitogenic action of estrone involved the participation of ER and MAPK transduction pathway activation. The presence of estradiol impaired the effect of estrone on cell proliferation and vasoactive production. These results suggest that estrone exhibits a remarkable biological action on endothelial cells, modulating vasoactive production, proliferation, apoptosis, and cell adhesion events.

A randomized, multiple-dose parallel study to compare the pharmacokinetic parameters of synthetic conjugated estrogens, A, administered as oral tablet or vaginal cream.

OBJECTIVE: A randomized, parallel-design study was conducted to determine the pharmacokinetic profile of synthetic conjugated estrogens A (SCE-A) vaginal cream (0.625 mg SCE-A/g) when administered at intervals (1 g once daily for 7 d, then twice weekly) over a 27-day period as compared with the pharmacokinetic profile of 0.3 mg SCE-A tablets administered once daily orally for 27 days. METHODS: Blood samples were collected 48 hours before initial dosing for baseline levels and at multiple occasions during the study until 48 hours after final study dosing (day 29). Maximum plasma concentration, time to maximum plasma concentration (Tmax), and area under the curve from 0 to 24 hours were calculated at days 1, 7, and 27; in addition, area under the curve from 0 to 48 hours was calculated at days 7 and 27, and area under the curve weekly (AUCweekly) values were calculated for both groups. For purposes of comparison, ratios of AUCweekly values for vaginal cream and oral tablets were analyzed. RESULTS: Compared with an oral daily dose of 0.3 mg SCE-A, the steady-state systemic exposure from vaginal cream application was considerably less, with the cream-to-oral ratio being 0.45 for baseline-adjusted (BA) unconjugated estradiol, 0.30 for BA unconjugated estrone, and 0.04 for unconjugated equilin (AUCweekly). At steady-state, the systemic blood levels of BA unconjugated estrone, BA unconjugated estradiol and unconjugated equilin were significantly lower in women who received biweekly application of 1 gm vaginal cream compared to women who took an oral daily dose of 0.3 mg SCE-A tablet. CONCLUSIONS: After intravaginal application of SCE-A vaginal cream, absorption of estrogens was lower compared with absorption after oral administration. At steady state, the systemic exposure of equilin, estradiol, and estrone was significantly lower after twice-weekly administration of 1 g SCE-A vaginal cream compared with that achieved with an oral daily dose of a 0.3 mg SCE-A tablet.

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.

NMR and computational studies of stereoisomeric equine estrogen-derived DNA cytidine adducts in oligonucleotide duplexes: opposite orientations of diastereomeric forms.

The equine estrogens equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive o-quinone form in aerated aqueous solutions. The o-quinones react predominantly with C, and to a lesser extent with A and G, to form premutagenic cyclic covalent DNA adducts in vitro and in vivo. To obtain insights into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically modified oligonucleotides containing the stereoisomeric 1'S,2'R,3'R-4-OHEN-C3 and 1'R,2'S,3'S-4-OHEN-C4 adducts derived from the reaction of 4-OHEN with the C in the oligonucleotide 5'-GGTAGCGATGG in aqueous solution. A combined NMR and computational approach was utilized to determine the conformational characteristics of the two major 4-OHEN-C3 and 4-OHEN-C4 stereoisomeric adducts formed in this oligonucleotide hybridized with its complementary strand. In both cases, the modified C adopts an anti glycosidic bond conformation; the equilenin distal ring protrudes into the minor groove while its two proximal hydroxyl groups are exposed on the major groove side of the DNA duplex. The bulky 4-OHEN-C adduct distorts the duplex within the central GC*G portion, but Watson-Crick pairing is maintained adjacent to C* in both stereoisomeric adducts. For the 4-OHEN-C3 adduct, the equilenin rings are oriented toward the 5'-end of the modified strand, while in 4-OHEN-C4 the equilenin is 3'-directed. Correspondingly, the distortions of the double-helical structures are more pronounced on the 5'- or the 3'-side of the lesion, respectively. These differences in stereoisomeric adduct conformations may play a role in the processing of these lesions in cellular environments.