Hepatic Estrogen Receptor Research Articles

17β-Estradiol treatment increases the levels of estrogen receptor and its mRNA in male rat liver

This study examined estrogen receptor dynamics in the livers of male obses rats ( SHHF Mcc-cp ) treated for two weeks with a continuous, low dose of 17β-estradiol compared with untreated controls. An increased binding capacity for tritiated 17β-estradiol in the cytosol, consistent with binding to the estrogen receptor, was demonstrated in treated males relative to control males ( P < 0.01). These observations were confirmed using curve-peeling techniques with saturation analysis, ammonium sulfate precipitation/fractionation of cytosol protein, and chromatographic techniques to isolate the high-affinity binding from other interfering factors. Increased hepatic nuclear estrogen receptor levels in treated males (112.3 ± 8.3 fmol/g liver) compared with controls (64.1 ± 6.8 fmol/g liver) suggested that the liver was under estrogenic influences. This interpretation was supported by an increase in serum triglyceride levels, reflecting increased very low density lipoprotein secretion by the liver. Reductions in testosterone levels and in the weights of seminal vesicles and the testes in treated males indicated detrimental effects on reproduction. An interpretation of increased synthesis of estrogen receptor with 17β-estradiol treatment was supported by the observation of an increase in the mRNA for estrogen receptor. Taken together, these observations indicate that continuous, low-dose 17β-estradiol treatment induces estrogenic action in the livers of male rats and also increases hepatic estrogen receptor, probably indirectly, via an increase in its mRNA.

Characterization of protein interactions with positive and negative elements regulating the apoVLDLII gene.

Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is estrogen dependent and liver specific. Competence to express the apoVLDLII gene is not acquired until days 7-9 of embryogenesis and thus lags 5-6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to hepatic estrogen receptor profiles, or a requirement for other transcription factors not expressed at earlier stages of embryogenesis. The latter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrogen administration. We have examined the influence of these hypersensitive regions on expression from the apoVLDLII promoter and have characterized novel protein-DNA interactions at two of them. One is located in a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predicted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotides downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G residues contacted by these proteins decreases hormone-inducible expression from the promoter 5- to 8-fold. Hepatic levels of the liver-enriched factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determinant of the ability to express the apoVLDLII and possibly other liver-specific genes.

The hormonal regulation of the oestrogen receptor in rat liver: an interplay involving growth hormone, thyroid hormones and glucocorticoids

The regulation of the formation of the hepatic oestrogen receptor (ER) in adult female rats was studied by assaying steady state levels of ER and ER messenger RNA under different endocrine conditions. Hypophysectomy (HX) drastically reduced ER levels from 67.5 +/- 7.9 to 8.4 +/- 0.5 (means +/- S.E.M.) fmol/mg cytosolic protein. Continuous infusion of growth hormone (GH) to HX animals tripled ER and doubled ER mRNA levels. Treatment with triiodothyronine (T3) in a high dose (10 micrograms/day) doubled ER mRNA levels. The effects of T3 were dose-dependent, since a lower dose (1 microgram/day) increased neither ER nor ER mRNA levels. ER mRNA concentrations were increased by GH to 481 +/- 44% and by T3 to 372 +/- 35% of HX control levels 4 h after single injections of the hormones in HX animals. The glucocorticoid dexamethasone (DEX) alone increased neither ER nor ER mRNA levels in HX animals. DEX and GH in combination increased ER 5-fold and ER mRNA 2-fold compared with control levels in HX animals, whereas DEX and T3 in combination increased neither ER nor ER mRNA levels. Treatment with prolactin affected neither ER nor ER mRNA levels in HX rats. Insulin-like growth factor I (IGF-I) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured. GAPDH mRNA levels were increased 2.5-fold in HX rats by DEX and T3 in combination and almost 2-fold by DEX and GH in combination. IGF-I mRNA levels in HX rats were increased 4.5-fold by continuous infusion of GH alone, 6-fold by GH and T3 in combination, and 2.5-fold by GH and DEX in combination. These data indicate that both GH and T3 act directly on the liver to increase ER mRNA levels. GH, the most important of these hormones, also acts at the translational and/or post-translational level to increase ER protein levels. DEX treatment suppresses the stimulatory effects of T3, but not of GH.

Estrogen regulation of the estrogen receptor and insulinlike growth factor-I in the rat uterus: a potential coupling between effects of estrogen and IGF-I

The interrelationship between estrogen and insulinlike growth factor-I (IGF-I) in the regulation of uterine growth was studied in the rat. The levels of the estrogen receptor (ER), ER mRNA, and IGF-I mRNA in rat uterus and liver monitored. Uterine ER in normal cycling rats highest in proestrus and diestrus, as was IGF-I mRNA. ER and mRNA and plasma estradiol peaked in proestrus. Hepatic ER and mRNA and IGE-I mRNA were highest in diestrus, whereas ER was not significantly changed suring the estrous cycle. The temporal effects of multiple infections or continuous infusion of 17 β-estradiol in ovariectomized rats were examined. In the uterus of animals subjected to multiple injections, a 10-fold increase in IGF-I mRNA was seen 24 h after the start of the treatment, whereas rats given continuous infusion estradiol showed a more than 16-fold increase. In both groups, the inrease of IGF-I mRNA was transient although estrogen treatment was continued. To study local hormonal effects, ovariectomized rats were given estradiol in vaginal implants. The uterine IGF-I mRNA level increased two-fold in 3 days. The ER and mRNA level increased 1.5-fold and the uterine weights were doubled. The plasma estradio concentration did not change during the treatment. A separate experiment was carried out to establish whether IGF-I itself exercises estrogenlike effects.Ovariectomized rats were given hrIGF-I in osmotic minipumps for 3 days. The uteri of the treated animals weighed significantly more than did the controls. Quantitation of the level of uterine estrogen receptors revealed a significant decrease. In conclusion, estrogen appears to be an important regulator of IGF-I mRNA since an increase in IGF-I mRNA covaries with estrogen during the estrous cycle. In ovariectomized rats, systematic administration of estrogen transiently increased IGF-I mRNA. Administration of IGF-I can cause estrogenic effects on the uterus in terms of increased weight. IGF-I down-regulated the uterine estrogen receptor, which suggests a reciprocal dependence between estrogen and IGF-I in the effects on the uterus.

Estrogen action on hepatic synthesis of angiotensinogen and IGF-I: Direct and indirect estrogen effects

In the present study effects of estrogens (natural estradiol and synthetic ethinyl estradiol) on liver derived proteins (angiotensinogen, IGF-I) were investigated in vivo in ovariectomized rats and in vitro in a rat hepatoma cell line (Fe33). The aim of this study was to establish both an animal and an in vitro model for quantification of the hepatic activity of given estrogenic compounds, and to study underlying mechanisms as regards the question of direct or indirect mode of estrogen action. In ovariectomized rats subcutaneous (s.c.)-treatment for 11 days with either estradiol (E2) or ethinyl estradiol (EE) (dose range 0.1–3 μg/animal/day) induced a comparable dose-dependent increase in uterine weight indicating a similar estrogenic potency of the two estrogens. Equipotency was also found as regards the effects on IGF-I plasma levels which dose-dependently decreased by about 50% at the highest dose tested (3 μg/animal/day). The decrease in IGF-I serum levels was accompanied by a significant 40% decrease in liver IGF-I mRNA. In contrast angiotensinogen plasma levels were affected only by EE (60% increase for the 3 μg/animal/day dose) but not by E2. When rats, in addition to ovariectomy, were also hypophysectomized (substituted with human growth hormone and dexamethasone) angiotensinogen again increased by 80% upon administration of 3 μg/animal/day EE, whereas IGF-I remained unaffected by EE. In a rat hepatoma cell line (Fe33) which is stably transfected with an estrogen receptor expression plasmid, 10 nmol/1 EE for 24 h caused a 2.4-fold increase in angiotensinogen mRNA level. We conclude from our studies that estrogen effects on angiotensinogen serum levels in the rat are direct effects via the hepatic estrogen receptor, whereas estrogen effects on IGF-I serum levels are indirect effects, the primary target of estrogen action being probably the pituitary. The changes in angiotensinogen serum levels in the rat model are comparable to the situation in humans indicating the rat model and the Fe33 model to be useful tools to study the hepatic activity of estrogenic compounds.

The influence of ethanol and liver disease on sex hormones and hepatic estrogen receptors in women

Clinical signs of sex hormone disturbances in men with alcoholism and/or liver diseases consist of testicular atrophy, hypo- or aspermia, sexual dysfunction, gynecomastia, and loss of axillary hair. Disturbances of the menstrual pattern in women with cirrhosis as well as in alcoholic women have been recognized for many years. However, alcoholic patients have other medical problems, for example liver and/or pancreatic dysfunction, malnutrition and/or infectious diseases that can contribute to endocrine disturbances. In comparison to the numerous studies of sex hormone changes in men with chronic liver diseases, very few studies have addressed the question of sexual, menstrual or sex hormone changes in women with liver diseases. Whether the sex hormone disturbances observed in alcoholic patients are consequences of liver dysfunction or induced by ethanol or both is still debated.

Induction of the estrogen receptor by growth hormone and glucocorticoid substitution in primary cultures of rat hepatocytes

Hepatic estrogen receptors (ER) mediate estrogenic effects on mammalian liver metabolism and are thereby involved in the regulation of important physiological/pathological processes, such as coagulation, atherosclerosis, and hypertension. The regulation of the formation of the ER in primary cultures of rat hepatocytes was studied by assaying ER and ER mRNA under different endocrine conditions. The ER concentration was measured using two different methods, a ligand-binding technique and an ER enzyme immunoassay. The results obtained by the two methods showed good correlation, and linear regression analysis gave a correlation coefficient of 0.95. ER concentrations fell to low steady state levels within 16 h after establishing the cell culture and remained low in the absence of hormonal substitution. Upon medium supplementation with pituitary GH and the glucocorticoid dexamethasone (DEX) in combination, the ER concentration increased 6-fold from 4.2 +/- 1.0 to 25.8 +/- 7.0 fmol/mg cytosolic protein. ER mRNA was measured by solution hybridization. Substitution with GH and DEX in combination increased ER mRNA to 210 +/- 14% of control levels. No effect on ER mRNA stability was seen after hormone treatment. It is concluded that the regulatory effects of GH and DEX on the hepatic ER in this in vitro system are very similar to the effects of these hormones under in vivo conditions. The inducible expression of the ER has never before, to our knowledge, been demonstrated in any mammalian liver cell culture system.

Induction of the estrogen receptor by growth hormone and glucocorticoid substitution in primary cultures of rat hepatocytes.

Hepatic estrogen receptors (ER) mediate estrogenic effects on mammalian liver metabolism and are thereby involved in the regulation of important physiological/pathological processes, such as coagulation, atherosclerosis, and hypertension. The regulation of the formation of the ER in primary cultures of rat hepatocytes was studied by assaying ER and ER mRNA under different endocrine conditions. The ER concentration was measured using two different methods, a ligand-binding technique and an ER enzyme immunoassay. The results obtained by the two methods showed good correlation, and linear regression analysis gave a correlation coefficient of 0.95. ER concentrations fell to low steady state levels within 16 h after establishing the cell culture and remained low in the absence of hormonal substitution. Upon medium supplementation with pituitary GH and the glucocorticoid dexamethasone (DEX) in combination, the ER concentration increased 6-fold from 4.2 +/- 1.0 to 25.8 +/- 7.0 fmol/mg cytosolic protein. ER mRNA was measured by solution hybridization. Substitution with GH and DEX in combination increased ER mRNA to 210 +/- 14% of control levels. No effect on ER mRNA stability was seen after hormone treatment. It is concluded that the regulatory effects of GH and DEX on the hepatic ER in this in vitro system are very similar to the effects of these hormones under in vivo conditions. The inducible expression of the ER has never before, to our knowledge, been demonstrated in any mammalian liver cell culture system.

Binding of xenobiotics to the estrogen receptor of spotted seatrout: A screening assay for potential estrogenic effects

Several xenobiotics that bind to mammalian estrogen receptors (ER) were tested for their ability to bind to the hepatic ER of a marine fish, the spotted seatrout ( Cynoscion nebulosus). Incubation of cytosolic extracts with the antiestrogens clomiphene, tamoxifen, and nafoxidine caused displacement of 3H-estradiol from its receptor. Kepone (chlordecone) also bound to seatrout ER, but it had a lower affinity than that observed with mammalian ER. Preliminary results indicated that these compounds have antiestrogenic or estrogenic actions in the spotted seatrout. However, several DDT derivatives and PCB mixtures failed to displace estradiol from the spotted seatrout ER, even though they have been shown to bind to mammalian ER. The lack of binding to spotted seatrout ER by several of the xenobiotics may be due to structural differences between ER in spotted seatrout and in mammals. These results indicate that the spotted seatrout ER assay can be used for screening xenobiotics for possible estrogenic activity in teleosts.

Endocrine mechanisms for the formation of sex-related differences in hepatic estrogen receptor content and their significance for the realization of an estrogen effect on angiotensinogen blood level in rats.

The use of a modified, adequate method of quantification of estrogen receptors has permitted us to prove the existence of sex-specific peculiarities in rat liver estrogen reception and their significance for the realization of sex-dependent changes in angiotensinogen plasma level after estrogenization. Endocrine mechanisms for the formation of sex-related differences in hepatic estrogen receptor content in rats were investigated in detail. The investigation shows that androgens have negative regulatory influence on the hepatic estrogen receptor level in rats. Estrogens and adrenal and thyroid hormones do not take part in the regulation of hepatic estrogen receptor content in rats. It has been proven that the decisive role in keeping up a certain estrogen receptor concentration in hepatocytes belongs to pituitary growth hormone. It was shown for the first time that androgens are able to inhibit the stimulatory effect of growth hormone on hepatic estrogen receptors.

Antiestrogenic action of 2,3,7,8-tetrachlorodibenzo- p-dioxin: Tissue-specific regulation of estrogen receptor in CD1 mice

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is a polychlorinated aromatic hydrocarbon with teratogenic and carcinogenic properties. Previous studies in our and other laboratories have demonstrated that TCDD has antiestrogenic properties. In order to elucidate the mechanism of action of TCDD on estrogen sensitive tissues, we studied its effects on serum estradiol and estrogen receptor (ER) levels in liver and uteri of CD1 mice. Treatment with TCDD did not result in alterations of serum estradiol levels at any of the doses tested (1.0–30 μg/kg). In contrast, TCDD treatment induced a dose-dependent decrease in hepatic and uterine ER protein as determined by an enzyme immunoassay and equilibrium binding assays. A decrease in cytosolic and nuclear ER levels in uteri occurred as early as 24 hr after initial treatment with 30 μg/kg TCDD and recovery occurred by 14 days. Hepatic cytosolic and nuclear ER also decreased at a dose of 30 μg/kg TCDD at 24 hr after treatment, but recovery occurred only by 21 days. Studies in ovariectomized mice indicate that the regulation of hepatic ER by TCDD is independent of ovarian factors, but ovariectomy inhibited the downregulation of uterine ER by TCDD. Furthermore, determination of TCDD-induced cytochrome P-450 levels indicates that the downregulation of uterine ER is uncoupled from induction of hepatic cytochrome P-450. This study indicates that the antiestrogenic effects of low doses of TCDD are mediated through its ability to decrease hepatic and uterine ER and are not due to alterations in serum estradiol levels. Our results on ovariectomized mice indicate that TCDD-induced downregulation of ER is tissue specific and may involve different mechanisms at transcriptional or posttranscriptional levels.

Variation in hepatic estrogen receptor concentrations in patients with liver disease. A multivariate analysis.

Hepatic estrogen receptor concentrations were measured in liver biopsy specimens from 102 patients (58 women and 44 men) with liver disease and correlated to several clinical, biochemical, and histologic background variables by means of multiple regression analysis. Half of the tissue was processed for histologic evaluation with a semiquantitative registration of histologic characteristics, whereas the other part was used for receptor analysis by enzyme immunoassay. Fifteen patients with no or minimal histologic changes served as controls. The analysis shows that the reduced estrogen receptor concentrations observed in patients with chronic liver diseases reflect the degree of liver dysfunction and not the specific type of liver disease. Serum albumin, log serum bilirubin, log serum alkaline phosphatases, and the degree of parenchymal fibrosis were significantly related to hepatic estrogen receptor level in the final regression model, but only part of the variation can be explained by these variables, and other factors must be of importance.

Quantitation of estrogen and androgen receptors in hepatocellular carcinoma and adjacent normal human liver.

Sex hormones have been shown to influence the development and course of several liver diseases. The worldwide predominance of hepatocellular carcinoma (HCC) in males has led to the suggestion that this disease might be hormone-responsive. Therefore, the hepatic estrogen (ER) and androgen receptor (AR) status of liver specimens from such patients was investigated. Samples were obtained from three female and six males patients undergoing liver resection; in each case, a small sample of both the tumor and adjacent normal tissue was collected. All patients had primary hepatocellular carcinoma without cirrhosis. In most cases, the tumor and the normal specimen had an equivalent content of cytosolic ER; however, three of the tumor samples (one female and two male) displayed considerably elevated cytosolic ER levels as compared to that of the normal tissue. In every sample, the tumor contained less nuclear ER than did the normal liver. When AR was measured, tumors of three patients (one female and two male) demonstrated a twofold elevation in cytosolic AR as compared to adjacent normal tissue. In the two male patients, an approximately twofold greater nuclear AR was found. Two other samples from male patients showed a modest elevation of cytosolic AR in the tumors. The patients whose tumors showed elevations in ER were not the same patients as those in whom the AR was elevated. Thus, these studies indicate that certain, but not all, specimens of HCC demonstrate either elevated ER or AR and suggest that a determination of receptor content might be useful prior to initiation of certain antihormone therapies.

The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the hepatic estrogen and glucocorticoid receptors in congenic strains of Ah responsive and Ah nonresponsive C57BL/6J mice

The present study examines the role of the Ah receptor in the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the binding capacity of the hepatic glucocorticoid (GC) and estrogen (E) receptors in female congenic C57BL/6J mice differing only at the Ah (aromatic hydrocarbon responsiveness) locus. The Ah locus is thought to encode the Ah receptor, which regulates the effects of TCDD and related compounds on cytochrome P450IA1 and appears to mediate most of the toxic effects of TCDD. The differences between Ah responsive (Ah b/b) and nonresponsive ( Ah d d ) mice appear to reflect differences in the affinity of the Ah receptor in the two strains for ligands such as TCDD. Administration of a single oral dose of TCDD (30 μg/kg) to Ah b/b mice produced approximately a 30% decrease in the maximum binding capacities of both the hepatic GC and E receptors, as well as a 50-fold induction of a P450IA1-mediated enzymatic activity, ethoxyresorufin- O-deethylase (EROD). Tyrosine aminotransferase (TAT) activity, which is mediated by the GC receptor, was also decreased ∼30% by TCDD. Dose-response curves indicated that Ah responsive mice are 10-fold more sensitive to induction of EROD than Ah nonresponsive mice (ED50 1.6 vs 15 μg/kg), as would be expected for an effect mediated by the Ah receptor. Dose-response curves also indicated that there was a statistical difference in the responsiveness of the hepatic E receptor to TCDD in the two congenic strains of mice ( p < 0.01). Surprisingly, no significant differences in the dose-response curves for the effect of TCDD on hepatic GC receptor binding or TAT activity were observed in the two strains of mice in two separate experiments. These results indicate that the Ah receptor regulates the effects of TCDD on the binding of estrogen to the hepatic estrogen receptor, but suggest that the decrease in the binding capacity of the hepatic GC receptor does not appear to be mediated directly by the Ah locus.

In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout

We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E 2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.

Changes in hepatic estrogen-receptor concentrations during the annual reproductive and ovarian cycles of a marine teleost, the spotted seatrout, Cynoscion nebulosus

The concentrations of estrogen and vitellogenin in plasma, and hepatic estrogen receptor in cytosolic (ER c) and nuclear (ER n) extracts were elevated throughout the reproductive season in females from a wild population of spotted seatrout, Cynoscion nebulosus. There were significant correlations between plasma estradiol and ER c and between ER n and plasma vitellogenin during the period of ovarian recrudescence (January–May), but not during the remainder of the reproductive season (June–September). During ovarian recrudescence ER c concentrations increased fourfold from 0.78 ± 0.15 pmol/g liver ( N = 26) in nonvitellogenic females to 3.23 ± 0.26 pmol/g liver ( N = 77) in late vitellogenic females and ER n concentrations increased eightfold from 0.16 ± 0.07 pmol/g liver ( N = 26) in nonvitellogenic females to 1.12 ± 0.45 pmol/g ( N = 10) in mid vitellogenic females. However, following this period of ovarian recrudescence, estrogen-receptor concentrations and plasma vitellogenin titers did not fluctuate during the remaining stages of the ovulatory cycle (hydration, ovulation, and spawning). In addition, both hepatic ER c and ER n were elevated for the rest of the seasonal reproductive cycle, during which several ovulatory cycles may have occurred in this multiple-spawning species. The affinity of ER c for estradiol did not vary ( K d = 1.26 ± 0.06 n M, N = 68) regardless of the stage of ovarian development. Plasma estradiol titers declined in August, near the end of the reproductive season, prior to the decline in estrogen-receptor concentrations. The persistence of the estrogen receptor during the ovulatory cycle suggests that even if plasma estradiol titers declined between successive ovulatory periods, the hepatic responsiveness to estrogenic stimuli would not be diminished and thus vitellogenin synthesis, if interrupted at all, could be resumed soon after spawning.

Effects of chronic ethanol intake on aromatization of androgens and concentration of estrogen and androgen receptors in rat liver

The present study was carried out to investigate if ethanol alters aromatization of androgens and concentrations of hepatic estrogen and androgen receptors. Hepatic aromatization of androgen to estrogen was significantly increased by ethanol administration. There was a significant increase in serum estrogen level but a decreased circulating testosterone level in alcohol-fed rats. Furthermore, the concentration of estrogen receptors in liver cytosol was significantly higher in alcohol-fed rats (37 ± 5.3 fmol/mg protein), as compared to the intact control value (21 ± 4.8 fmol/mg protein). However, hepatic androgen receptor levels were much lower (4.4 ± 0.5) in alcohol-fed rats than those (10.2 ± 1.4 fmol/mg protein) in control animals. Similarly, castration increased hepatic aromatization of androgens and concentrations of serum estrogen and hepatic estrogen receptors, but it decreased contents of circulating androgen and hepatic androgen receptors. These findings indicate that alcohol administration is considered a chemical form of castration, altering the hepatic steroid metabolism and sex hormone receptor contents and contributing to the pathogenesis of ferminization. A combination of alcohol-feeding and castration has no synergistic effect on the hepatic steroid receptors and aromatization, but this combination does have a more profound effect in lowering the concentration of circulating androgen.

In vivo and in vitro estimations of the direct effect of estrogen on rat hepatocytes tested by the changes in the unusual estrogen-binding protein content

The direct effect of estradiol (E 2) on the hepatocytes of mature male rats has been examined by measuring the changes in the unusual estrogen-binding protein (UEBP) content and parallel measuring the level of liver estrogen receptors (ER). The content of UEBP ( N UEBP) and ER ( N ER) in the liver were determined using the quantitative methods for differential specific determination of the E 2-binding sites of these proteins. It has been shown that the administration of E 2 in vivo induced a considerable decrease in hepatic N UEBP not only in intact males, but also in hypophysectomized males during the initial period after the operation (when the content of hepatic ER was still high) and produced no effect in hypohysectomized males during the later period (when liver ER were depleted). Repeated administration of human growth hormone (hGH) (twice a day) resulted in a considerable increase in n er in hypophysectomized males and restored the sensitivity to the subsequent inhibitory effect of E 2 on UEBP. We also used rat hepatocytes after a 4-day primary culturing. These cells had a stable morpho-functional status, high ER level, and sex-differentiated UEBP content. Culturing of mature male rat hepatocytes in the medium containing E 2 at concentrations close to physiological levels (10 −10−10 −7) decreased N UEBP in a dose-dependent manner. Hexestrol (10 −7M) but not cholesterol (10 −5M) also exhibited a direct effect on N UEBP in cultured rat hepatocytes. The effect of E 2 was reversible: statistically significant increase in N UEBP was observed 3 days after 10 −9 M E; had been removed from the culturing medium. It was concluded that hepatocytes may be a primary target for E 2 under physiological conditions and that GH may modulate the direct effect of E 2 at the hepatic level by modifying the content of liver ER.

Binding characteristics of the hepatic estrogen receptor of the spotted seatrout, Cynoscion nebulosus

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of adult female spotted seatrout, Cynoscion nebulosus. A single class of high affinity binding sites was found, K d = 1.26 ± 0.55 n M ( N = 55) for the cytosolic extract and K d = 1.96 ± 0.42 n M ( N = 8) for the nuclear extract. The K d did not differ between males and females or between vitellogenic and nonvitellogenic females. The binding in both the cytosolic and nuclear extracts was specific for estrogens (DES > E 2 > > E 1 = E 3). Receptor concentrations in cytosolic extracts from late vitellogenic females (14.61 ± 1.07 pmol/g liver, N = 40) were significantly higher than those from nonvitellogenic females (3.91 ± 0.73 pmol/g liver, N = 7). The nuclear binding capacity of livers from midvitellogenic females (1.12 ± 0.45 pmol/g liver, N = 10) was significantly higher than the binding capacity in livers from nonvitellogenic females (0.16 ± 0.07 pmol/g liver, N = 26), but not that of late vitellogenic females (0.80 ± 0.09 pmol/g liver, N = 77). The concentration of estradiol-binding sites was greatest in the liver (liver > > ovary > heart > spleen > muscle > brain). No interference from other steroid-binding proteins was detected using a simple dextran-coated charcoal method to separate bound from free hormone. Approximately 14% of the binding in the cytosolic extract had DNA-binding affinity. Estrogen receptor binding activity was maximally extracted from nuclei with buffer containing 0.6 M KCI. Nuclear receptors eluted from gel filtration columns with an apparent molecular weight of 95 kDa.

Effect of antiandrogen flutamide on measures of hepatic regeneration in rats.

Male rat liver undergoes a process of demasculinization during hepatic regeneration following partial hepatectomy. The possibility that antiandrogens might potentiate this demasculinization process and in so doing augment the hepatic regenerative response was investigated. Adult male Wistar rats were treated with the antiandrogen flutamide (2 mg/rat/day or 5 mg/rat/day subcutaneously) or vehicle for three days prior to and daily after a 70% partial hepatectomy. At various times after hepatectomy, the liver remnants were removed and weighed. Rates of DNA and polyamine synthesis were assessed by measuring thymidine kinase and ornithine decarboxylase activities, respectively. Hepatic estrogen receptor status and the activity of alcohol dehydrogenase, an androgen-sensitive protein, were measured. Prior to surgery, the administration of 5 mg/day flutamide reduced the hepatic cytosolic androgen receptor activity by 98% and hepatic cytosolic estrogen receptor content by 92% compared to that present in vehicle-treated controls. After hepatectomy, however, all differences in sex hormone receptor activity between the treatment groups were abolished. The rate of liver growth after partial hepatectomy in the three groups was identical. Moreover, hepatectomy-induced increases in ornithine decarboxylase activity and thymidine kinase activity were comparable. These data demonstrate that, although flutamide administration initially alters the sex hormone receptor status of the liver, these affects have no effect on the hepatic regenerative response following a partial hepatectomy.