The stereochemical complementarity of DNA and reproductive steroid hormones correlates with biological activity

Modeling studies revealed that progesterone, testosterone, and estradiol are stereochemically complementary to the cavity formed between base pairs in the DNA sequence, 5'-dTdG-3' X 5'-dCdA-3'. Each steroid aligned precisely with the topography of the cavity and formed 2 stereospecific hydrogen bonds linking phosphate oxygens on adjacent DNA strands. Hydrogen bonding donor-acceptor relationships were different for each hormone. The remarkable stereochemical specificity of the hormone-DNA complexes was demonstrated by the lack of complementarity of steroid enantiomers and steroid analogs having alternate ring systems and/or changes in the position of functional groups. Fit of molecules into DNA in the manner of the parent hormone correlated with biological activity. Antagonists also fit into the cavity but differed from agonists in their hydrogen bonding linkages to DNA and/or extended out of the cavity beyond the helix. Unlike flat intercalating agents which form stable complexes with DNA, wedge shaped steroids may thus be capable of forming reversible sequence-specific complexes with DNA. We conclude that the stereochemistry of DNA can be used to predict hormonal activity.     

The stereochemistry of hydrogen transfer to NADP+ by enzymes acting upon stereoisomeric substrates

The stereochemistry of hydrogen transfer from estradiol-17α and estradiol-17β to NADP⁺ in the presence of chicken liver estradiol-17α and estradiol-17β dehydrogenases was found in both cases to involve the 4-pro-S proton of the pyridine nucleotide. One of these enzymes must therefore use the stereochemically less favorable mode of interaction of steroid with coenzyme.     

Catechol estradiol induced implantation in the mouse

Induction of implantation is among the most sensitive responses to estrogens. The ability of catechol estradiols, 4-hydroxy-estradiol-17 beta (4-OH-E2) and 2-hydroxy-estradiol-17 beta (2-OH-E2), to induce implantation in ovariectomized pregnant mice was compared to that of estradiol-17 beta. Delayed implantation was maintained by the daily administration of 2 mg of progesterone. A single injection of 3 ng of estradiol-17 beta, 50 ng of 4-OH-E2, or 2,000 ng of 2-OH-32 consistently induce a full complement of implantation sites in all animals. Before the estrogenicity of the latter steroid can be established the lack of contaminating estrogens must be proved.     

In vitro aromatic bioactivation of the weak estrogen E(2)alpha and genesis of DNA adducts

Specific A-ring hydroxylated metabolites of 17beta-estrogens are known to be endogenous pro-carcinogens, more particularly the 4-hydroxylated forms of estrogens produced by cytochrome P4501B1. In this study, we investigated whether estradiol-17alpha, the main hepatic residue of estradiol-17beta in cattle treated for anabolic purposes with estradiol containing implants, could be significantly metabolized by human cells, and whether its aromatic metabolites could induce the formation of DNA adducts as estradiol-17beta and estrone do. First, using a human kidney adenocarcinoma cell line, which expresses specifically the cytochrome P4501B1, we showed that estradiol-17alpha is bioactivated into a mixture of 2- and 4-catechol estrogens leading to the corresponding methoxyestrogens unambiguously identified by LC-APCI-MS/MS. We then demonstrated that the 2- and 4-hydroxylated derivatives of estradiol-17alpha incubated under oxidative conditions with calf thymus DNA gave stable DNA adducts and abasic sites, respectively. From these results, we can consider that human cells expressing CYP1B1-dependent hydroxylation activities metabolize estradiol-17alpha at the same magnitude as estradiol-17beta and estrone, and that in oxidative conditions, the resulting aromatic metabolites can lead to the formation of both stable and unstable DNA adducts.     

Placental and luteal steroidogenesis in the pregnant rhesus monkey.

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.     

Estrogen receptors and growth response in cultured human periodontal ligament cells

The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning. PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta. Using [3H]estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein. With [3H]dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein. With [3H]R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein. Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor. The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively. The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth. These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens.     

Steroid metabolism by human mammary carcinoma.

The ability of human mammary tumors to convert 7alpha 3H-testerone to estrogens was examined in order to determine whether this bore any relationship to estrogen receptor and steroid sulfurylation levels; such levels being indicative of hormone dependency. In 8 out of 9 tumors, formation of estradiol-17beta from testosterone was demonstrated. Those tumors showing the lowest conversion of testosterone to estradiol-17beta possessed the highest levels of dehydroepiandrosterone sulfotransferase which lends support to data implicating sulfurylation in the regulation of steroid metabolism in human tumors. All tumors activated sulfate to adenosine-3'-phospho-5'-phosphosulfate and the concentrations were significantly correlated withe the recorded levels of dehydroepiandrosterone sulfate. Estrogen receptor levels did not show any obvious relationship to the other parameters.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

Adduction of catechol estrogens to nucleosides

We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the catechol estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium.In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.     

Crystal structure of a recombinant anti-estradiol Fab fragment in complex with 17beta -estradiol.

The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Studies on the cross stand hydrogen bonds in DNA double helices.

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross-strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may in fact be considered as yet another general structural feature of DNA helices.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Conversion of steroids in bovine blood in vitro

Blood samples collected from eight Braunvieh cows between the sixth and eighth month of gestation were allowed to stand with and without anticoagulant at 20 degrees C and 0 degrees C for different time periods. In these samples the degree of in vitro conversion of gestagens, androgens and estrogens was investigated. The concentrations were measured by radioimmunoassay. After 24 h at 20 degrees C, the levels of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone and estrone decreased to 62, 29, 25, 10, 34 and 44%, respectively, of the initial value and those of 17alpha, 20beta-dihydroxyprogesterone, epitestosterone and estradiol-17alpha increased to 385, 800 and 852%, respectively. The conversion was slower in clotted blood. The concentrations of testosterone and estradiol-17beta were consistent over the 24 h period. There was no marked decrease of the steroid concentration after 24 h of incubation of whole blood at 0 degrees C and of plasma at 20 degrees C. After the addition of (3)H-steroids, conversion could be demonstrated by thin-layer chromatography and autoradiography. These results demonstrate that all investigated hormones except testosterone and estradiol-17beta were metabolized by bovine blood cells.     

Studies On The Cross Strand Hydrogen Bonds In DNA Double Helices

Abstract

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross- strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may infact be considered as yet another general structural feature of DNA helices.     

Hydrogen bonds in protein-DNA complexes: where geometry meets plasticity

Recognition of a DNA sequence by a protein is achieved by interface-coupled chemical and shape complementation. This complementation between the two molecules is clearly directional and is determined by the specific chemical contacts including mainly hydrogen bonds. Directionality is an instrumental property of hydrogen bonding as it influences molecular conformations, which also affects DNA-protein recognition. The prominent elements in the recognition of a particular DNA sequence by a protein are the hydrogen-bond donors and acceptors of the base pairs into the grooves of the DNA that must interact with complementary moieties of the protein partner. Protein side chains make most of the crucial contacts through bidentate and complex hydrogen-bonding interactions with DNA base edges hence conferring remarkable specificity.     

A double antibody radioimmunoassay for free and conjugated estradiol-17 beta in cow's milk

A radioimmunoassay for free estradiol-17 beta, conjugated estradiol-17 beta or total (free + conjugated) estradiol-17 beta in defatted milk of cows is described. Conjugated estradiol-17 beta was hydrolyzed by enzymes of Helix pomatia juice. Estrogens were extracted with dichloromethane; no other purification step was required before radioimmunoassay because of the high specificity of the antiserum. Immunoprecipitation was used to separate bound and free estradiol-17 beta. Concentrations measured were corrected for procedural losses on a per sample basis. The assays were shown to be accurate and specific. The sensitivity was 1.3pg/ml for the assay of free estradiol-17 beta (5ml of milk extracted) and 2.9pg/ml for conjugated or total estradiol-17 beta (2 ml of milk hydrolyzed and extracted). Estrogens were measured in the milk of cyclic cows and in cows stimulated with pregnant mare serum gonadotropin (PMSG). A preovulatory increase was clearly observed. Wether or not the ovary was stimulated by PMSG, concentrations of estrogens were higher and the relative increase during the preovulatory peak was greater for conjugated estradiol-17 beta than for the free form. The assay of conjugated or total estradiol-17 beta in defatted milk should be a practical method for assessing preovulatory growth of follicles in cows.     

The solubilisation of some steroids by phosphatidylcholine and phosphatidylcholine-cholesterol vesicles.

The solubility of the three steroid hormones, progesterone, testosterone, and estradiol-17 beta in water and phosphatidylcholine vesicles was measured after shaking and ultrasonication. All three steroids have low water solubility, which increases considerably at sonication for testosterone and estradiol-17 beta. The phosphatidylcholine vesicles have a very small solubilising capacity for the steroids; about 20 mumol/mol. This increases at sonication for estradiol-17 beta and decreases for testosterone. The capacity for progesterone is almost unaltered. The incorporation of cholesterol in the vesicles decreased the solubilisation capacity for testosterone and estradiol-17 beta but increased that for progesterone of shaked preparations. For the sonicated systems the cholesterol decreased the solubilising capacity for estradiol-17 beta but increased that for testosterone. The solubilisation experiments indicate that the steroid hormones are solubilised in the hydrocarbon part of the phosphatidylcholine bilayer and also 13CNMR results support this conclusion.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.