Different Classes Of Steroids Research Articles

Effects of environmental steroid mixtures are regulated by individual steroid receptor signaling

Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRβ) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17β-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.

Development of Methods to Quantify Free and Conjugated Steroids in Fatty Matrices by HPLC-MS/MS.

Steroids are called the “fingerprint” of oils, fats, and their derivatives. Different classes of steroids may be present in these matrices. Most of the methods developed to analyze these constituents involve the determination of free steroid content, although their conjugated forms are extremely important in determining the total composition. Thus, this article demonstrates that the coupling of sequential mass spectrometry and high-performance liquid chromatography obtained high sensitivity and high specificity of mass resolution to identify and quantify the main classes of steroids. Four methods were developed to quantify steroids free, esterified, glucosides, and acylated glucosides by internal standardization using betulin. The main validation parameters were tested and demonstrated good correlation results for the methods. The content of free steroids was the majority in all samples, whereas the content of glucoside steroids was the least abundant. The contents of free steroids quantified in the degummed soybean oil were significantly reduced in relation to the refined oil. A small amount of esterified steroids was superior in refined soybean oil than in degummed oil. Comparing the steroid content between degummed oil and biodiesel, we found that the concentration of free and esterified steroids decreases in the conversion to biodiesel, whereas the concentration of glucoside steroids increases slightly.

Physiological and Transcriptional Effects of Mixtures of Environmental Estrogens, Androgens, Progestins, and Glucocorticoids in Zebrafish

Fishes are exposed to mixtures of different classes of steroids, but ecotoxicological implications are not sufficiently known. Here, we systematically analyze effects of different combinations of steroid mixtures in zebrafish embryos to assess their joint activities on physiology and transcriptional alterations of steroid-specific target genes at 96 and 120 h post fertilization. In binary mixtures of clobetasol propionate (CLO) with estradiol (E2) or androstenedione (A4), each steroid exhibited its own expression profile. This was also the case in mixtures of 5-, 8-, and 13-different classes of steroids in exposure concentrations of 10-10,000 ng/L. The transcriptional expression of most genes in different mixtures was steroid-specific except for genes encoding aromatase (cyp19b), sulfotransferase (sult2st3), and cyp2k22 that were induced by androgens, progestins, and glucocorticoids. Marked alterations occurred for sult2st3 in binary mixtures of CLO + E2 and CLO + A4. Glucocorticoids increased the heart rate and muscle contractions. In mixtures containing estrogens, induction of the cyp19b transcript occurred at 10 ng/L and protc from the anticoagulation system at 100 ng/L. Our study demonstrates that steroids can act independently in mixtures; the sum of individual steroid profiles is expressed. However, some genes, including cyp19b, sult2st3, and cyp2k22, are regulated by several steroids. This joint effect on different pathways may be of concern for fish development.

Interaction of environmental steroids with organic anion transporting polypeptide (Oatp1d1) in zebrafish (Danio rerio).

Steroid hormones in the aquatic environment may pose a risk to fish health due to their ubiquitous presence and high biological activity. At present, the uptake process and toxicokinetics of steroids in fish are poorly known, in particular the role of cell membrane transporters. We investigated the interaction of 17 endogenous and environmental steroids with the zebrafish organic anion transporting peptide (Oatp1d1) uptake transporter, which is prominently expressed in liver and kidneys. We selected steroids of different classes including androstenedione (A4), progesterone (P4), and its metabolites, as well as glucocorticoids and spironolactone, and analyzed their interaction with Oatp1d1 by competitive inhibition of the uptake of the fluorescent substrate Lucifer Yellow. The half-maximal inhibition (IC50) values derived from sigmoid inhibition curves were lowest for P4, and the order of increasing IC50 values was as follows: 17α-hydroxyprogesterone > clobetasol propionate > spironolactone > 21α-hydroxyprogesterone > fludrocortisone acetate and additional glucocorticoids. The interaction activity showed a positive correlation with the lipophilicity of the steroids. Our data show that different classes of steroids interact with Oatp1d1 with different activity (either by uptake or inhibition, or both). This is of importance, because in consequence, steroids may interfere with the transport of endogenous substrates, and thus physiological processes. Moreover, steroids may alter cellular trafficking of environmental contaminants by competitive inhibition of this transporter. Environ Toxicol Chem 2018;37:2670-2676. © 2018 SETAC.

Determination of two progestin metabolites (17α-hydroxypregnanolone and pregnanediol) and different classes of steroids (androgens, estrogens, corticosteroids, progestins) in rivers and wastewaters by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

A highly sensitive and robust method was developed for routine analysis of two progestin metabolites, 17α-hydroxypregnanolone (17OH-Δ5P) and pregnanediol (PD), and 31 other natural and synthetic steroids and related metabolites (estrogens, androgens, corticosteroids, progestins) in river water, as well as influents and effluents of municipal wastewater treatment plants (WWTP) using HPLC-MS/MS combined with solid-phase extraction. For the various matrixes considered, the optimized method showed satisfactory performance with recoveries of 70–120% for most of target steroids. The method detection limits (MDLs) ranged from 0.01 to 3ng/L for river water, 0.02 to 10ng/L for WWTP effluents, and 0.1 to 40ng/L for influents with good linearity and reproducibility. The developed method was successfully applied for the analysis of steroids in rivers and WWTP influent and effluents. WWTP influents concentrations of 17OH-Δ5P and PD were 51–256ng/L and up to 400ng/L, respectively, along with androstenedione (concentration range: 38–220ng/L), testosterone (11–26ng/L), estrone (2.3–37ng/L), 17β-estradiol (N.D.–8.7ng/L), 17α-hydroxyprogesterone (N.D.–66ng/L), medroxyprogesterone acetate (N.D.–5.3ng/L), and progesterone (2.0–22ng/L), while only androstenedione (ADD), estrone (E1), and estriol (E3) were detected in effluent with concentrations ranging up to 1.7ng/L, 0.90ng/L and 0.8ng/L, respectively. In river water samples, only ADD and E1 were detected with concentrations up to 1.0ng/L and 0.91ng/L. Our procedure represents the first method for analyzing 17OH-Δ5P and PD in environmental samples along with a large series of steroids.

Steroid bioaccumulation profiles in typical freshwater aquaculture environments of South China and their human health risks via fish consumption

More attention was previously paid to adverse effects of steroids on aquatic organisms and their ecological risks to the aquatic environment. So far, little information has been reported on the bioaccumulative characteristics of different classes of steroids in cultured fish tissues. The present study for the first time provided a comprehensive analysis of the occurrence, bioaccumulation, and global consumers’ health risks via fish consumption of androgens, glucocorticoids and progestanges in typical freshwater cultured farms in South China. The numbers and total concentrations of steroids detected in the tissues of five common species of the cultured fish were in the order of plasma > bile > liver > muscle and plasma > bile, muscle > liver, respectively. The field bioaccumulation factors for the detected synthetic steroids ranged from 450 to 97,000 in bile, 450 to 65,000 in plasma, 2900 to 16,000 in liver, and 42 to 2600 in muscle of fish, respectively. This data suggests that steroids are bioaccumulative in fish tissues. Mostly important, 4-androstene-3,17-dione (AED) and cortisone (CRN) were found to be reliable chemical indicators to predict the levels of steroids in plasma and muscle of the inter-species cultured fish, respectively. Furthermore, the maximum hazard quotients (HQs) of testosterone and progesterone were 5.8 × 10−4 and 9.9 × 10−5, suggesting that human health risks were negligible via ingestion of the steroids-contaminated fish.

Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids

The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9α-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshA(DSM43269) homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshA(DSM43269) homologues are C-9 α-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 α-hydroxylation occurring at different levels during microbial steroid degradation.

Transcription factors as regulators of steroidogenic P-450 enzymes.

Steroid hormones are essential for normal sexual development, accommodation to stress, and regulation of fluid and electrolyte balance. Biosynthesis of these different classes of steroids and its appropriate regulation requires the precisely controlled expression of six different cytochrome P-450 enzymes and two hydroxysteroid dehydrogenases in different tissues. The molecular mechanism of the tissue-specific and pituitary hormone-regulated expression of the genes encoding P-450 enzymes in the steroidogenic tissues is the central problem of long-term regulation of steroidogenesis. Orphan members of the nuclear receptor superfamily play an important role in mediating transcriptional regulation of several steroid hydroxylase genes. Two of these transcription factors, steroidogenic factor-1 (SF-1) and DAX-1, will be reviewed here in detail.

Determinants of Ligand Specificity of Estrogen Receptor-α: Estrogen versus Androgen Discrimination

We have been interested in understanding how the estrogen receptor (ER) binds estrogens and discriminates between different classes of steroids with closely related structures. Using insights from our prior studies on ER and from sequence comparisons of steroid receptors, we identified three residues in the hormone-binding domain of the human ER, Leu345, Thr347, and Glu353, that we considered were likely to be involved in steroid A-ring recognition and therefore estrogen versus androgen discrimination. We then tested the effect on ER activity of mutating these ER residues to the corresponding androgen receptor residues. Specifically, we examined the ability of the mutant receptors to bind and be activated by 17beta-estradiol and three different androgens. No change in receptor activity was observed with the T347N mutation, while the L345S mutation greatly reduced ER activity in response to all ligands. Interestingly, the E353Q substitution behaved as expected, causing a 9-fold reduction in the transactivation potency of estradiol and a concomitant 10-140-fold increase in the transactivation potency of different androgens. These reciprocal changes in the transcriptional effectiveness of estrogens and androgens correlated with a decreased affinity of the E353Q ER for estradiol binding and an increased affinity for androgen binding. Therefore, amino acid Glu353 appears to be playing a significant role in binding the A-ring phenolic group of estradiol and in receptor discrimination between estrogens and the most closely structurally related steroids, androgens. Based on this data and our earlier observations, we propose a model for the orientation of ligand within the binding pocket of ER in which the A-ring 3-phenol of estradiol is hydrogen bonded to Glu353 in helix-3 and the 17beta-hydroxyl of estradiol is hydrogen bonded to His524 in helix-11. Our findings with estrogen and androgen suggest that this orientation of the steroid in the ligand-binding pocket, with the steroid A-ring in contact with helix-3 and the D-ring in contact with helix-11 residues, is likely to be general for all the steroid hormone receptors.

Expression and characterization of a novel UDP-glucuronosyltransferase, UGT2B9, from cynomolgus monkey.

Uridine diphosphate glucuronosyltransferases (UGTs) are important phase II detoxification enzymes. Despite the expression of UGT proteins in many species, previous results have suggested that simians represent the most appropriate animal model to study the glucuronidation of steroids in extrahepatic steroid target tissues. Northern blot analysis using a pool of human UGT2B cDNA probes demonstrated the expression of homologous UGT2B transcripts in several tissues including the liver, kidney, adrenal, breast, testis, and prostate of the cynomolgus monkey (Macacafascicularis). Western blot analyses using a polyclonal antibody raised against human UGT2B17 protein also demonstrated expression of homologous UGT2B proteins in monkey tissues. cDNA libraries were constructed from monkey liver and prostate mRNA and a novel UGT2B cDNA, UGT2B9, was isolated from both libraries. The UGT2B cDNA from the prostate library is 2,648 bp in length and contains an open reading frame of 1,587 bp encoding a protein of 529 residues. In vitro transcription/translation of the cDNA clone produced a protein of 52 kD. The UGT2B9 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B9 protein was established. The activity of UGT2B9 was tested with over 60 compounds and was demonstrated to be active on C18, C19, and C21 steroids, bile acids, and several xenobiotics including eugenol, 1-naphthol, and p-nitrophenol. Kinetic analysis revealed that UGT2B9 glucuronidates steroids with high affinity and efficiency with Km values of 0.2, 3.2, 0.2, and 1.8 microM for dihydrotestosterone, testosterone, androsterone, and 1,3,5,10-estratrien-3,4-diol-17-one, respectively. It is apparent that this simian UGT2B enzyme is specific for more different classes of steroids than any other UGT enzyme characterized to date, and may be related to the high plasma levels of glucuronidated C19 steroids found in the cynomolgus monkey.

The two faces of a steroid antagonist: When an antagonist isn't

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function. The recent documentation of conditional agonist activity in antagonists of several different classes of steroids could have significant implications for the use of steroid antagonists in the clinical setting, representing a previously unrecognized mechanism for the development of steroid resistance.

17O NMR spectroscopic investigation of steroids at natural abundance

AbstractNatural abundance 17O NMR spectroscopic data, in toluene‐d8 at 90°C, were obtained for different classes of steroids, including androstanes, estranes, pregnanes, cholanes and cholestanes. Only routine acquisition parameters were used in combination with linear prediction methods during the processing for eliminating acoustic ringing artifacts. The results are discussed in terms of stereochemical effects on the 17O chemical shift of the various oxygen‐containing functionalities.

Specific effect of corticoids on acetylcholine receptor expression in rat skeletal muscle cell cultures.

The potential effect of different classes of steroids on the expression of acetylcholine receptors (AChR) was studied in different primary cultures of newborn-rat skeletal muscle cells. Comparison among three techniques for preparing newborn skeletal muscle cells showed that these systems were equivalent to study AChR expression. Only corticoids stimulated myogenesis as a twofold increase in AChR expression indicated. Among the corticoids, the glucocorticoids were the more potent, whereas the mineralocorticoid aldosterone had less marked effect. The sex hormones progesterone and testosterone partially blocked these effects, without inducing any significant effect when given alone. The steroids tested differed in efficacy in correlation with their different chemical structures. Among the glucocorticoids a clear structure-activity relationship could be established. These results emphasize the specificity of corticoid action on muscle cells and suggest an explanation for the effects induced by glucocorticoids used in treating human muscular or neuromuscular diseases.

Effect of different classes of steroids on myogenesis of cultured rat muscle cells

Effect of different classes of steroids on myogenesis of cultured rat muscle cells

On the steroid requirement for induction of tyrosine aminotransferase by N6, O2'-dibutyryl cyclic AMP in hepatoma cells.

Dibutyryl cyclic AMP induces tyrosine aminotransferase significantly in several lines of cultured cells derived from either normal rat liver or hepatomas even when glucocorticoids are not present in the culture medium. Preincubation of the cells with glucocorticoids, however, markedly augments the subsequent response to the cyclic nucleotide. The ability of different classes of steroids to promote induction of tyrosine aminotransferase by dibutyryl cyclic AMP was defined by incubation with glucocorticoid agonists and antagonists prior to the addition of dibutyryl cyclic AMP. Steroids with glucocorticoid activity enhance the induction of tyrosine aminotransferase by dibutyryl cyclic AMP, and yield an increased level of immunoreactive enzyme in the cells. Inactive steroids and glucocorticoid antagonists are ineffective in this regard. Thus, dexamethasone (an optimal agonist) and 21-deoxycortisol (a partial agonist) promote enzyme induction by dibutyryl cyclic AMP. Progesterone, testosterone and 17β-estradiol (glucocorticoid antagonists) and tetrahydrocortisol (an inactive agent) neither induce the enzyme themselves nor amplify the induction by dibutyryl cyclic AMP. Progesterone and other antagonists, however, inhibit the induction of tyrosine aminotransferase by dexamethasone alone or combined with dibutyryl cyclic AMP in both HTC and H-35 monolayer cultures. Glucocorticoids and cyclic AMP therefore act independently to induce tyrosine aminotransferase in the basal state, but also interact to produce a response that is much more than additive and requires the steroid receptor.

Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-forming cells to steroids of different classes

Selected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolane suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man.

Steroids and Hematopoiesis. III. The Response of Granulocytic and Erythroid Colony-forming Cells to Steroids of Different Classes

AbstractSelected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolone suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man.

Steroids and hematopoiesis II. The effect of steroids on in vitro erythroid colony growth: Evidence for different target cells for different classes of steroids

Androgenic steroids and their non-androgenic 5beta-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unit/ml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Preincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells. Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid. Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed. Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.