RESUMO
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.
Assuntos
Fracionamento Químico/métodos , Estrogênios/química , Equilenina/química , Equilina/análogos & derivados , Equilina/química , Estradiol/química , Estrona/química , Íons , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Estrogênios/química , Progestinas/química , Poluentes Químicos da Água/química , Equilina/análogos & derivados , Equilina/química , Estradiol/química , Estrona/química , Etinilestradiol/química , Modelos Químicos , Ozônio/químicaRESUMO
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.
Assuntos
Equilina/química , Estradiol/química , Estrona/química , Oxidantes/química , Poluentes Químicos da Água/química , Animais , Humanos , Radical Hidroxila/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Ozônio/químicaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Adutos de DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Envelhecimento , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Adutos de DNA/análise , Adutos de DNA/química , Equilenina/análogos & derivados , Equilenina/química , Equilenina/metabolismo , Equilina/análogos & derivados , Equilina/química , Equilina/metabolismo , Estrogênios Conjugados (USP)/administração & dosagem , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Estrogênios/química , Salinidade , Sonicação/métodos , Poluentes Químicos da Água/química , Água/química , Equilina/análogos & derivados , Equilina/química , Estradiol/química , Concentração de Íons de HidrogênioRESUMO
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.
Assuntos
Citidina/química , Adutos de DNA/química , Equilenina/análogos & derivados , Equilina/química , Congêneres do Estradiol/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Animais , Sequência de Bases , Citidina/metabolismo , Dano ao DNA , Equilenina/química , Equilenina/metabolismo , Equilina/metabolismo , Cavalos , Humanos , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , EstereoisomerismoRESUMO
Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum.
Assuntos
Estrogênios/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Esteroides/análise , Sulfonas/química , Calibragem , Cromatografia Líquida , Equilenina/análise , Equilenina/química , Equilina/análise , Equilina/química , Estradiol/análogos & derivados , Estradiol/sangue , Estradiol/química , Imidazóis/química , Padrões de Referência , Esteroides/químicaRESUMO
4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.
Assuntos
DNA Polimerase Dirigida por DNA/química , Equilenina/química , Equilina/química , Estrogênios/química , Animais , Sequência de Bases , Catálise , Adutos de DNA , Feminino , Terapia de Reposição Hormonal/métodos , Cavalos , Humanos , Cinética , Modelos Químicos , Dados de Sequência MolecularRESUMO
Estrogens, especially estradiol, have been shown to stimulate the proliferation of hormone-dependent types of breast cancer cells. 17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) enzyme catalyses the synthesis of the active female estrogen, estradiol and is thus an attractive target for structure-based ligand design for the prevention and control of breast tumour growth. In this study, the active site of 17beta-HSD1 has been reviewed, and three crystal structure complexes (estradiol/NADP+, equilin/NADP+, dehydroepiandrosterone) of 17beta-HSD1 have been selected to be analysed for de novo ligand design. The boundary surface, hydrophobic interactions and hydrogen bonding sites in the ligand binding domain for each ligand complex were analysed to create a comprehensive image of the active site.
Assuntos
Desidroepiandrosterona/química , Equilina/química , Estradiol Desidrogenases/química , Estradiol/química , Software , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Ligantes , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Adutos de DNA/química , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , Desoxicitidina/química , Equilenina/análogos & derivados , Equilenina/química , Equilina/análogos & derivados , Animais , Pareamento Incorreto de Bases/genética , DNA/química , DNA/genética , Adutos de DNA/genética , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA/genética , Desoxicitidina/genética , Equilenina/genética , Equilina/química , Equilina/genética , Cavalos , Humanos , Cinética , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estereoisomerismo , Moldes GenéticosRESUMO
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.
Assuntos
Equilina/farmacologia , Congêneres do Estradiol/farmacologia , Animais , Equilina/análogos & derivados , Equilina/síntese química , Equilina/química , Congêneres do Estradiol/administração & dosagem , Congêneres do Estradiol/síntese química , Congêneres do Estradiol/química , Feminino , Modelos Moleculares , Conformação Molecular , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Útero/anatomia & histologia , Útero/efeitos dos fármacosRESUMO
Recently, we investigated the inhibitory effects of 17 beta-estradiol and diethylstilbestrol on microtubule assembly, cytotoxicity, and aneuploidy in V79 cells. The present study analyzes the effects of equilin and equilenin (amongst the natural estrogens originally isolated from the urine of pregnant horses) and their related compounds, on the relative plating efficiency of Chinese hamster V79 cells. The results showed that a hydroxyl group on 17-C and a methoxyl group on 3-C of the estrogen skeleton were important for cytotoxicity. Of the various compounds analyzed, 2-methoxyestradiol had the strongest cytotoxicity, suggesting also the importance of a methoxyl group on 2-C.
Assuntos
Equilenina/toxicidade , Equilina/toxicidade , Estradiol/análogos & derivados , Estradiol/toxicidade , 2-Metoxiestradiol , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Equilenina/química , Equilina/química , Estradiol/química , Estrona/toxicidade , Relação Estrutura-Atividade , Testes de ToxicidadeRESUMO
3-Hydroxyestra-1,3,5(10),7-tetraen-17-one, C18H20O2, crystallizes in space group P2(1)2(1)2(1) from ethyl acetate. The planarity of the B ring, and the difference in puckering of the C and D rings from that of estrone, are due to the presence of the C7 = C8 double bond, which may explain its function as an inhibitor of human type 1 17 beta-hydroxysteroid dehydrogenase, instead of being its substrate.
Assuntos
Cristalografia por Raios X , Equilina/química , Terapia de Reposição de Estrogênios , Humanos , Modelos MolecularesRESUMO
Excess 17beta-estradiol (E2), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which catalyzes the reduction of inactive estrone (E1) to the active 17beta-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E2 in breast tumor tissues. We present here the structure of the ternary complex of 17beta-HSD1 with the cofactor NADP+ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17beta-HSD1-catalyzed reduction of E1 to E2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-A resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP+ and catalytic residues Tyr-155 and Ser-142 is different from that of E2 in the 17beta-HSD1-E2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17beta-HSD1 is the basis for inhibition of E1-to-E2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.
Assuntos
Equilina/metabolismo , Estradiol Desidrogenases/química , Estradiol Desidrogenases/metabolismo , NADP/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Equilina/química , Humanos , Análise dos Mínimos Quadrados , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , NADP/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
Underivatized estrone (ES), equilin (EQ), equilenin (EQN) and their corresponding 17 alpha-diols 17 alpha-estradiol (ESD), 17 alpha-dihydroequilin (DHEQ) and 17 alpha-dihydroequilenin (DHEQN) were separated by TLC, RP-HPLC and capillary GC. Their dipole moments (mu) and Randic's connectivity indices ((1)chi) were determined as parameters of importance for the separation. The number of H atoms was taken as an additive structural parameter of importance for the quantitative structure-chromatographic retention relationship study (QSRR). Principal component analysis (PCA) was applied in order to find similarities and dissimilarities between 9 TLC and 10 RP-HPLC systems. PCA indicated that proton donor-proton acceptor interactions play the most important role for the TLC and RP-HPLC separation. The two-dimensional non-linear map of PC variables showed that the keto-estrogens (ES, EQ and EQN) and the corresponding diols (ESD, DHEQ and DHEQN) form two separate clusters. The relationship between GC retention of equine estrogens characterized by Kováts indices (KI), their (1)chi and mu was expressed by the equation KI/100 = al(1)chi+ b/mu(2) + c. The biological activity of the estrogens was related to log 1/mu(2).