Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10^?9M for females and 1.7 × 10^?9M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K_D = 1.5 × 10^?9M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.     

Presence of three different estradiol binding proteins in rat prostate cytosol

Depending upon the experimental conditions, three distinct [³H]-17β-estradiol binding entities can be detected in rat prostate cytosol. A protein with characteristics similar to other estrogen receptors is found in prostate cytosol from intact rats. This protein has a sedimentation coefficient of 8S on sucrose gradients. It has a high affinity only for natural and synthetic estrogens. It decreases rapidly after castration (less than 20% of binding activity after 24 hr) and reappears progressively after 8–10 days.A second binding entity with a sedimentation coefficient of 4–5S on sucrose gradients is also observed. It is found both in intact and castrated rats. At low temperatures, the binding of estradiol increases progressively and reaches a maximum after 24–48 hr of incubation as determined by charcoal assay. This binding species is highly specific for natural estrogens but not for diethylstilbestrol and is probably identical with the 4–5S binder.Finally, a third binding entity is found in 24 hr castrated rats with short incubations at 0°C This binding is labile even at low temperatures. Natural and synthetic androgens compete more strongly than estradiol for this binding. This behaviour suggests that the third estradiol binding entity is identical with the androgen receptor.     

Estrogen interaction with the anterior pituitary of female rats differential cytosol binding, nuclear translocation and stimulation of RNA synthesis by 17β-estradiol and tamoxifen

The interaction of tamoxifen (trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene) with the cytosol estrogen receptor of the anterior pituitary of female rats was studied. No differences were recorded between incubations of cytosol samples with 17β-[³H]estradiol performed in the presence or absence of unlabeled 17β-estradiol and tamoxifen, respectively, thus suggesting that these interactions were at common receptor sites and excluding possible cooperative interactions. Competition experiments and Scatchard plot analysis of saturation experiments add further evidence for common receptor sites. A dissociation constant for tamoxifen of K_d = 2 nM was recorded. Tamoxifen was found to be bound to a moiety sedimenting in the 4–5 S region, on a 6–24% linear sucrose density gradient at low salt concentrations, whereas 17β-estradiol sedimented in the 8–9 S area. These data suggest possible conformational changes of the receptor in the presence of tamoxifen. Furthermore, nuclear estrogen receptor levels remained elevated for at least 80 h after the application of tamoxifen alone or in a combination with 17β-estradiol, and a concomitant inhibition of cytosol receptor replenishment was noted. Tamoxifen and 17β-estradiol, respectively, were found to stimulate progesterone receptor levels when applied through 5 days. Tamoxifen plus 17β-estradiol administration elevated progesterone receptor contents above those found for each of the two compounds alone. On the other hand, tamoxifen enhanced the 17β-estradiol-induced prolactin serum levels, but did not stimulate prolactin serum levels by itself. These data combine to suggest that tamoxifen interacts with common estrogen receptor sites at the rat anterior pituitary.     

Phosphorylation of calf uterus 17 beta-estradiol receptor by endogenous Ca2+-stimulated kinase activating the hormone binding of the receptor

Direct evidence is presented that uterus 17_β-estradiol receptor is phosphorylated $\text{in}\text{,}\text{vitro}$ by an endogenous kinase. Nuclear phosphatase, cytosol Ca²⁺-stimulated kinase (the former inactivating and the latter reactivating the hormone binding of the 17_β-estradiol receptor) and receptor were purified from calf uterus. 17_β-estradiol binding was inactivated by phosphatase, then reactivated by kinase in the presence of [_γ-³²P] ATP, Ca²⁺ and calmodulin, and the receptor was examined by various methods. The results of gel electrophoresis in non denaturating and denaturating conditions, and of centrifugation through sucrose gradients of receptor preincubated with monoclonal antibodies showed that the receptor is phosphorylated.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

17β-Estradiol protects against apoptosis induced by levofloxacin in rat nucleus pulposus cells by upregulating integrin α2β1

Levofloxacin has been reported to have cytotoxicity to chondrocytes in vitro. And 17β-estradiol has been widely studied for its protective effects against cell apoptosis. Based on apoptotic cell model induced by levofloxacin, the purpose of this study was to explore the mechanism by which 17β-estradiol protects rat nucleus pulposus cells from apoptosis. Inverted phase-contrast microscopy, flow cytometry, and caspase-3 activity assay were used to find that levofloxacin induced marked apoptosis, which was abolished by 17β-estradiol. Interestingly, estrogen receptor antagonist, ICI182780, and functional blocking antibody to α₂β₁ integrin, both prohibited the effect of 17β-estradiol. Simultaneously, levofloxacin decreased cellular binding ability to type II collagen, which was also reversed by 17β-estradiol. Furthermore, western blot and real-time quantitative PCR were used to find that integrin α₂β₁ was responsible for estrogen-dependent anti-apoptosis, which was time–response and dose–response effect. 17β-estradiol was proved for the first time to protect rat nucleus pulposus cells against levofloxacin-induced apoptosis by upregulating integrin α₂β₁ signal pathway.     

Estrogen receptor in the thymus of the castrated mice.

Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradiol-17 beta in the presence of excess unlabeled steroids showed a specific estrogenbinding 4 S protein with its binding capacity of 10(-14) moles/mg protein for males and 4 x 10(-15) moles/mg protein for females, respectively. The dissociation constant was of 4 x 10(-10) M for males and 3 x 10(-10) M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node. Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradiol-17 beta showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to 4 S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4 S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a specific estrogen receptor, the nature of which is in part protein and heat-labile.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.     

Synthesis and properties of some O-(2,2-dialkoxyethyl)glycolaldehydes

β-d-Mannosidase (β-d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-d-mannosidase, β-d-galactosidase, 2-acet-amido-2-deoxy-d-glucosidase, (1→4)-β-d-mannanase, and (1→4)-β-d-glucanase activities, almost devoid of α-d-galactosidase activity, and contaminated with <0.02% of β-d-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-d-manno-oligosaccharides of d.p. 3–5 (12.5mm). The initial rate of hydrolysis of (1→4)-linked β-d-manno-oligosaccharides of d.p. 2–5 and of reduced β-d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-d-mannopyranosides were readily hydrolysed. β-d-Mannobiose was hydrolysed at a rate ～25 times that of 6¹-α-d-galactosyl-β-d-mannobiose and 6³-α-d-galactosyl-β-d-mannotetraose, and at ～90 times the rate for β-d-mannobi-itol.     

Comparative studies of the 17β-estradiol receptors in rabbit liver,kidney and uterus

The cytosol 17β-estradiol receptors from rabbit kidney, liver and uterus, compared under identical experimental conditions, were similar in terms of their pH-activity profiles, dependence on incubation temperature, sensitivity to sulfhydryl reagents and steroid specificity. 17β-[³H]-Estradiol binding was saturable with all three tissues, having an apparent dissociation constant of 4 × 10⁻¹⁰ M. The binding of 17β-[³H]-estradiol in kidney, liver and uterus was inhibited by estrogens, including estrogen conjugates, but not by testosterone, progesterone or cortisol. The 17β-estradiol receptors of liver, kidney and uterus exhibited significant differences with respect to their Chromatographie behaviour on heparinSepharose. Furthermore, a comparison of their sucrose density gradient centrifugation patterns showed that the 17β-[³H]-estradiol-receptor complex of liver and kidney sedimented at 3-4 S in both low and high ionic strength media, while the uterine receptor sedimented at 7–8 S in low ionic strength media and at 4–5 S in high ionic strength media. When the liver and uterine cytosol fractions were combined the uterine receptor was altered and sedimented at 3–4 S in low ionic strength media.     

Lack of selective killing by steroids in normal and malignant cells

Using a colony formation assay, the cytotoxic effects of steroids and an anit-steroid on an established human breast tumor line and two human diploid fibroblast strains were studied. Experiments involving 17 a-estradiol, 17 β-estradiol, dexamethasone, cortisone, dihydrotestosterone, and the anti-estrogen Tamoxifen showed no killing at concentrations below 10^–7M following a 24-hour exposure to these agents. A maximum of 80% killing was observed at 10^–5M with dexamethasone in one fibroblast strain and at the same dose of 17 β-estradiol in the breast tumor line. The extent of killing observed is insufficient to account for many of the clinical remissions observed with steroid therapy. The data also suggest that at therapeutic doses, there is no selective killing of malignant cells by these agents.     

Regulation of K(ATP) channel by 17β-estradiol in pancreatic β-cells

ATP-sensitive potassium channels (K_ATP) regulate electrical activity and insulin secretion in pancreatic β-cells. When glucose concentration increases, the [ATP]/[ADP] ratio rises closing K_ATP channels, and the membrane potential depolarizes, triggering insulin secretion. This pivotal role of K_ATP channels is used not only by glucose but also by neurotransmitters, hormones and other physiological agents to modulate electrical and secretory β-cell response.In recent years, it has been demonstrated that estrogens and estrogen receptors are involved in glucose homeostasis, and that they can modulate the electrical activity and insulin secretion of pancreatic β-cells. The hormone 17β-estradiol (E2), at physiological levels, is implicated in maintaining normal insulin sensitivity for β-cell function. Long term exposure to E2 increases insulin content, insulin gene expression and insulin release via the estrogen receptor α (ERα), while rapid responses to E2 can regulate K_ATP channels increasing cGMP levels through the estrogen receptor β (ERβ) and type A guanylate cyclase receptor (GC-A). This review summarizes the main actions of 17β-estradiol on K_ATP channels and the subsequent insulin release in pancreatic β-cells.     

Estrogen modulation of osteoclast lysosomal enzyme secretion

Osteoclast-mediated bone resorption is accomplished by secretion of lysosomal proteases into an acidic extracellular compartment. We have previously demonstrated that avian osteoclasts and human osteoclast-like giant cell tumor cells respond in vitro to treatment with 17β-estradiol (17β-E₂) by decreased bone resorption activity. To better understand the mechanism by which this is accomplished, we have investigated the effects of 17β-E₂ treatment on lysosomal enzyme production and secretion by isolated avian osteoclasts and multinucleated cells from human giant cell tumors in vitro. Isolated cells were cultured with bone particles in the presence of either vehicle or steroid. The conditioned media and cells were harvested, and the levels of cathepsin B, cathepsin L, β-glucuronidase, lysozyme, and tartrate-resistant acid phosphatase (TRAP) activities were determined. There was a steroid dose-dependent decrease in secreted levels of these enzymes. Cell-associated levels of cathepsin L, β-glucuronidase, and lysozyme decreased, whereas cell-associated levels of cathepsin B and TRAP increased. These changes were measurable at 10^?10 M and maximal at 10^?8 M 17β-E₂. The changes were detectable at 4–18 h of treatment and increased through 24 h of treatment. The response was steroid specific, since the inactive estrogen isomer, 17β-E₂, failed to alter the activity levels. Moreover, the effects of 17β-E₂ were blocked when the cells were treated simultaneously with the estrogen antagonist ICI182–780 in conjunction with 17β-E₂. Human osteoclast-like cells obtained from giant cell tumors of bone responded similarly to estrogen with respect to cathepsin B, cathepsin L, and TRAP activities. However, secretion of β-glucuronidase and lysozyme were not altered by treatment with 10^?8 M 17β-E₂. These data indicate that estrogen effects on osteoclast resorption activity may be mediated by decreasing the secretion of cathepsin B, cathepsin L, and TRAP.     

Protein production and release by dispersed rat submandibular gland cells in vitro after adrenergic stimulation

Enzymatically dispersed cell aggregates were prepared from rat submandibular glands. Cells were responsive to α- and β-adrenergic agonists, as measured by net K⁺ release and radiolabeled protein secretion, respectively. Protein production by submandibular gland cells was constant during the 90 min experimental period. Specific agonist and antagonist experiments demonstrated that both α- and β-adrenergic receptor stimulation were required for maximum secretion of newly synthesized protein. Proteins were radiolabeled with [³⁵S] methionine and both soluble cell and secreted proteins examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A broad size range of newly synthesized proteins was detected (Mr～10⁴?5 × 10⁵). Adrenergic stimulation (1-epinephrine) specifically increased the secretion of certain radiolabeled proteins and, in addition, resulted in both cellular and secreted proteins with electrophoretic characteristics distinct from that of control preparations.     

Response of primary rabbit kidney proximal tubule cells to estrogens

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red–free medium. 17β-estradiol was observed to stimulate growth at dosages as low as 10⁻¹⁰ M. The growth stimulatory effects of 17β-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10⁻⁹ to 10⁻⁸ M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17β-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17β-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17β-estradiol. 17β-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures. J Cell Physiol 178:35–43, 1999. © 1999 Wiley-Liss, Inc.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Characterization of estrogen binding in the developing rat testis. Ontogeny of the testicular cytoplasmic estrogen receptor.

The properties and physical characteristics of a steroid binding component present in the immature (7 to 35 day) rat were investigated and found to be different from those of the 17 beta-estradiol receptor in the mature rat testis. These properties include a binding capacity of 483 fmol estradiol/mg protein, a Ka at equilibrium of 4.23 x 10(7)M-1, and broad steroid specificity as shown by interaction with several steroids; no binding was observed with diethylstilbestrol. The component, found in blood and several tissues including the testis, migrated as a 4.6S peak on sucrose gradients. This 4.6S component, which interacted with an anti-alphafetoprotein antiserum, decreased with age and was not detectable in the testis after day 21 or in the serum after day 25. These data suggest that this component is alphafetoprotein. Ontogenic appearance of the testicular cytoplasmic 17 beta-estradiol receptor in the developing rat was further elucidated. Sucrose gradient sedimentation analysis of cytosols revealed an 8S binding component that was first detectable at 23 days. Specific binding (fmol [3H]-estradiol/testis) was relatively low in neonates, rose to 59 fmol during the third week, and increased dramatically to 333 fmol at 7 weeks; binding rose only gradually after maturity. The receptor was tissue specific and steroid specificity studies demonstrated that only diethylstilbestrol and other estrogens were effective in competing with 17 beta-estradiol for binding sites. The Ka at equilibrium was determined as 3 x 10(10)M-1 and the binding sites were saturable in an in vitro system. The receptor did not interact with anti-alphafetoprotein antiserum as indicated by sucrose gradient studies. These data demonstrate the developmental appearance of the testicular cytoplasmic estradiol receptor in the immature rat.     

17β-hydroxysteroid dehydrogenase activity in cultured myometrial cells: Effect of serial subcultures

17β-Hydroxysteroid dehydrogenase (17β-SDH) activity was studied in culture ovine rayometrial cells. After primary culture, cells were routinely subcultured (every 7th day), seeded at 5–10⁵ cells per dish and grown in a medium with 2% of serum. 17β-SDH activity was measured by incubating intact cell monolayers with [³H]-estradiol (5·10⁻⁶) in serum-free medium. Metabolites were extracted from both cells and medium, and separated by thin-layer chromatography. 17β-SDH was expressed as total E i formed (cells + medium) in fmol/mg of protein as a function of time. 17β-SDH has an approximate K_m of 5–10⁻⁶ M. After 3 min of incubation, all measurable E₁ is within the cells; it is progressively released but after l h only 40% of E₁ is found in the medium. 17β-SDH decreases from day 2 to day 8 of each subculture, whereas total proteins increase. Subculture partially restores 17β-SDH activity so that it is always higher on day 2 of any subculture than on day 8 of the previous one. however a progressive decline occurs with successive subcultures. This decline parallels the slowing of cell growth and overall protein synthesis and probably reflects cell ageing.     

Cytosol and nuclear estrogen and progesterone receptors in the rabbit endocervix

This report describes the measurement of estrogen and progesterone receptors in cytosols and nuclear fractions from endocervical tissue components. Unoccupied cytosol estrogen receptor levels as determined by Scatchard analysis of [³H]-estradiol binding data indicated a single class of high affinity binding sites for the epithelial-stromal complex (K_D = 0.74 × 10⁻⁹ M). Binding was specific for estrogen (estradiol > estriol > estrone) and unaffected by desoxycorticosterone, dihydrotesterone and progesterone. Assays for total estrogen receptor verified that 71.6 ± 5.3% of this 8S estrogen receptor is in the epithelial-stromal complex while the remaining approximately 28% is localized in the stroma and fibromuscular wall, with the cells of the complex containing the highest receptor concentration. In 5-day pseudopregnant and ovariectomized rabbits compared to estrous rabbits there was a 50% decrease in the cytosol estrogen receptor in the epithelial-stromal complex and a 30% decrease in the concentration of nuclear receptor. Cytosol and nuclear progesterone receptors were measured as an indicator of estrogen action in the rabbit endocervix. Cytosol progesterone receptor concentrations (fmol/mg DNA) in 5-day pseudopregnant and ovariectomized animals were reduced to approximately 35% of the concentration in estrous animals. Nuclear progesterone receptor concentrations decreased 65% in 5-day pseudopregnant and 90% in ovariectomized animals suggesting decreased receptor synthesis. Collectively these data support the concept that the rabbit endocervix may be directly regulated by estrogens.     

Oxysterol Regulation of Estrogen Receptor α-Mediated Gene Expression in a Transcriptional Activation Assay System Using HeLa Cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor α-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17β-estradiol >> genistein >> β-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = β-epoxysitosterol = 7β-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.