Structure/function analyses of human sex hormone-binding globulin: effects of zinc on steroid-binding specificity

In humans, sex hormone-binding globulin (SHBG) binds and transports the biologically most important androgens and estrogens in the blood, and regulates the access of these steroids to their targets tissues. In addition to binding sex steroids, SHBG has specific binding sites for divalent cations including calcium and zinc. Zinc binding to a site at the entrance of the steroid-binding pocket in human SHBG has been shown to reduce its affinity for estrogens, while having no impact on the binding of C19 steroids. Crystallographic studies indicate that C18 and C19 steroids are bound in opposite orientations within the SHBG steroid-binding site, and we have obtained new information that supports a molecular model explaining the mechanism by which zinc alters the affinity of human SHBG for estrogens, by studying directly the estradiol-binding properties SHBG variants created by site-directed mutagenesis. In this model, the coordination of a zinc ion by the side chains of residues Asp65 and His136 eliminates a critical hydrogen bond between Asp65 and the hydroxyl at C3 of estrogens, such as estradiol and 2-methoxyestradiol, and causes disorder in a polypeptide loop segment that covers the steroid-binding site. The combination of these structural changes explains the specific decrease in the affinity of human SHBG for C18 steroids in the presence of a zinc ion.     

Role of endocytosis in cellular uptake of sex steroids

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.     

Endocrine Disruption: Computational Perspectives on Human Sex Hormone-Binding Globulin and Phthalate Plasticizers

Phthalates are a class of high volume production chemicals used as plasticizers for household and industrial use. Several members of this chemical family have endocrine disrupting activity. Owing to ubiquitous environmental distribution and exposure of human population at all stages of life, phthalate contamination is a continuous global public health problem. Clinical and experimental studies have indicated that several phthalates are associated with adverse effects on development and function of human and animal systems especially the reproductive system and exposures during pregnancy and early childhood are by far of utmost concern. Sex hormone-binding globulin (SHBG) is a plasma carrier protein that binds androgens and estrogens and represents a potential target for phthalate endocrine disruptor function in the body. In the present study, the binding mechanism of the nine phthalates i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with human SHBG was delineated by molecular docking simulation. Docking complexes of the nine phthalates displayed interactions with 15–31 amino acid residues of SHBG and a commonality of 55–95% interacting residues between natural ligand of SHBG, dihydrotestosterone, and the nine phthalate compounds was observed. The binding affinity values were more negative for long chain phthalates DEHP, DNOP, DINP, and DIDP compared to short chain phthalates such as DMP and DBP. The Dock score and Glide score values were also higher for long chain phthalates compared to short chain phthalates. Hence, overlapping of interacting amino acid residues between phthalate compounds and natural ligand, dihydrotestosterone, suggested potential disrupting activity of phthalates in the endocrine homeostasis function of SHBG, with long chain phthalates expected to be more potent than the short chain phthalates.     

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Relationship between progesterone receptor binding and progestin biological activity

The binding affinities of a series of steroidal compounds for the hamster uterine progesterone receptor were determined using two sets of incubation conditions. These competitive binding conditions were designed to deduce the relative rates of ligand dissociation from the progesterone receptor. The progestin activity of these compounds was also determined in a bioassay employing the measurement of diamine oxidase in the traumatized hamster uterus. Steroids could be classified into two categories based on either an increase or decrease in relative binding affinity (RBA) with increasing time of competitive incubation. The mean (+/- SEM) progestin biopotency for the compounds having an increase in RBA was 120 +/- 18 (progesterone = 100), while the biopotency for compounds having a decrease in RBA was only 44 +/- 17. This difference was significant (P less than 0.01). Linear regression analyses revealed significant correlations between the RBAs and progestin biopotencies. Compounds showing a decrease in RBA with increasing time of incubation did not have antiprogestin activity. Kinetic studies of this type should be useful for selecting compounds with potent agonistic activity, but cannot unequivocally predict antihormonal activity.     

The unusual estrogen-binding protein (UEBP) of male rat liver: structural determinants of ligands

The unusual estrogen-binding protein (UEBP) found in a male rat liver is a sex dependent protein which differs from other known receptor and transport proteins by the high lability of its complexes with estradiol (E2) and also the unique specificity of affinity for hormones. In this work values of relative binding affinity (RBA) of the UEBP for 57 steroids and their analogs were determined. The affinity of steroids was characterised by the amount of the unlabeled compound needed for 50% inhibition of [3H]-E2 binding with the UEBP. A number of derivatives of estrane and androstane possess an ability to interact with this protein, in contrast to the derivatives of pregnane, stilbene and triphenylethane. Characterized by RBA values, natural steroids are found to have the following order: estriol larger than or equal to E2 greater than 16 alpha-hydroxyestrone = 2 alpha-hydroxytestosterone greater than 16-epiestriol greater than or equal to estetrol greater than or equal to 17-epiestriol greater than or equal to 2-methoxyestradiol greater than or equal to 5 alpha-androstane-3 alpha,17 beta-diol greater than or equal to estrone greater than testosterone greater than or equal to 2 beta-hydroxytestosterone greater than 5 alpha-dihydrotestosterone. Affinity of estrogens and androgens for the UEBP diminishes abruptly after removal of 3- and 17-hydroxy groups, masking of these by ether bonds or changing of 17 beta-hydroxyl to 17 alpha. All the investigated 17 oxo-C19-steroids, 5 beta-derivatives of testosterone, its 6 beta- and 16 alpha-hydroxy metabolites as well as 5 alpha-androstane-3 beta,17 beta-diol and 19-nortestosterone exhibit no essential affinity for the protein. On the basis of the results obtained it is suggested that the binding sites for estrogens and androgens in the UEBP molecule overlap but do not completely coincide.     

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.)

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.     

Influence of new hydroxylated triphenylethylene (TPE) derivatives on estradiol binding to uterine cytosol

Twelve homologous triphenyl acrylonitrile derivatives with a p-OH or p-CH3 group on one or more of the phenyl rings were synthesized in order to assess the relative influence of each position on binding to the estrogen receptor (ER) and on inhibition of prostaglandin synthetase (PGS). Their relative binding affinities (RBAs) for [3H]estradiol (E2)-labeled ER were compared at 0 and 25 degrees C in mouse and rat uterus cytosol with those of tamoxifen derivatives, cyclofenil and diethylstilbestrol. RBAs in both species were closely correlated (r = 0.92) although the RBAs were about twice as high in the mouse as in the rat. The unsubstituted skeleton had an RBA of much less than 0.1 (estradiol = 100). An OH-group in R1 or R2 (Fig. 1) engendered very low affinity whereas an OH-group in R gave rise to a compound with an RBA equivalent to that of E2, emphasizing the importance of this position in the interaction with ER. Compounds with an additional OH-group in R1 or R2 were significantly better competitors than E2. No further increase in RBA was noted with the trihydroxy derivative. The effect of the introduction of a hydrophobic CH3-group decreased affinity as expected in R, but also in position R1 unless a second OH-group was present in R2. None of the 12 test-compounds competed significantly for binding to the "anti-estrogen binding site" in rat kidney supernatant. Although polar groups were not necessary for inhibition of PGS, inhibition was enhanced by the presence of a hydroxy group in R or R1 (but not R2). Even greater inhibition was obtained by the further introduction of a CH3-group in R1 or R respectively. The conformations of these derivatives are compared to those of known estrogen ligands and anti-inflammatory agents in order to obtain further information on these protein recognition sites.     

(Arene)Cl₂Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: interaction with sex hormone binding globulin and anticancer activity

(Arene)dichloridoruthenium(II) complexes with N-coordinated isonicotinates of androgens (6) and estrogens (9) were prepared and tested for affinity to the estrogen receptor (ERα) and sex hormone binding globulin (SHBG), as well as for cytotoxicity in cancer cells. None of the new complexes bound noticeably to the ER and most of them also bound less strongly to SHBG than the corresponding unmetallated steroids 7. In MTT assays the Ru(p-cymene) complexes 9 of 2-substituted estrones were equally or even more cytotoxic than the metal-free steroids against hormone-dependent (MCF-7 breast and KB-V1 cervix carcinomas) and hormone-independent (518A2 melanoma) cells. The addition of external SHBG to MTT assays lowered the cytotoxicities of the complexes 9 and distinctly more so those of some steroids 7, probably by the way of sequestration and reduction of the cellular uptake. In the absence of SHBG the estrogen complexes 9 were internalized by 518A2 melanoma cells and ruthenated their DNA as quantified by ICP-OES. They also ruthenated salmon sperm DNA but did not change the topology of plasmid DNA in EMSA experiments. In addition, the Ru(p-cymene) complex of 2-ethoxyestrone (9c) was shown to reduce the motility of 518A2 melanoma cells in a wound-healing assay.     

Diverse roles for sex hormone-binding globulin in reproduction

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in blood and regulates their access to target tissues. Hepatic production of SHBG fluctuates throughout the life cycle and is influenced primarily by metabolic and hormonal factors. Genetic differences also contribute to interindividual variations in plasma SHBG levels. In addition to controlling the plasma distribution, metabolic clearance, and bioavailability of sex steroids, SHBG accumulates in the extravascular compartments of some tissues and in the cytoplasm of specific epithelial cells, where it exerts novel effects on androgen and estrogen action. In mammals, the gene-encoding SHBG is expressed primarily in the liver but also at low levels in other tissues, including the testis. In subprimate species, Shbg expression in Sertoli cells is under the control of follicle-stimulating hormone and produces the androgen-binding protein that influences androgen actions in the seminiferous tubules and epididymis. In humans, the SHBG gene is not expressed in Sertoli cells, but its expression in germ cells produces an SHBG isoform that accumulates in the acrosome. In fish, Shbg is produced by the liver but has a unique function in the gill as a portal for natural steroids and xenobiotics, including synthetic steroids. However, salmon have retained a second, poorly conserved Shbg gene that is expressed only in ovary, muscle, and gill and that likely exerts specialized functions in these tissues. The present review compares the production and functions of SHBG in different species and its diverse effects on reproduction.     

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.     

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.     

Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG).

This study reports on some environmental chemicals with estrogenic activity (xenoestrogens) and their binding interaction for human plasma sex-hormone binding globulin (hSHBG). The binding affinity constant of these xenoestrogens was measured in equilibrium conditions by solid phase binding assay, and their ability to displace endogenous testosterone and estradiol from hSHBG binding sites was determined with an ammonium sulfate precipitation assay in native plasma from normal men and women. The data showed that some of these xenoestrogens bind hSHBG, with a reversible and competitive binding activity for both [3H]testosterone and [3H]17beta-estradiol and with no apparent decrease in the number of hSHBG binding sites. Their respective binding affinity constants were low, ranging from 0.02 to 7.8 10(5) 1 x mol(-1). However, in native plasma from normal men and women, they were able to dose-dependently increase concentrations of hSHBG-unbound testosterone and/or estradiol. In this study, 4-nonylphenol and 4-tertoctylphenol, two alkylphenols used as surfactants in many commercial products, and bisphenol A and O-hydroxybiphenyl, widely used in the plastics industry, were identified as potent hSHBG-ligands. Additionally, the flavonoid phytoestrogens genistein and naringenin were also identified as hSHBG ligands, whereas their glucoside derivatives, genistin and naringin, had no binding activity for hSHBG. From these data, it is suggested that hSHBG binding may transport some contaminant xenoestrogens into the plasma and modulate their bioavailability to cell tissues. On the other hand, xenoestrogens may also displace endogenous sex steroid hormones from hSHBG binding sites and disrupt the androgen-to-estrogen balance. Whether xenoestrogen SHBG ligands could reach high enough concentrations in the blood to expose humans to any such effect merits further investigation.