The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver.

Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [3H]estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.     

Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate

Specific estrogen-binding sites have been demonstrated in purified nuclear fractions of prostates from intact rats. Saturation analysis of nuclei over a wide range of [³H]-estradiol concentrations (0.15 to 90 nM) has shown two different types of binding sites: a) one with high affinity (Kd of 0.5–0.8 nM) and low capacity for estradiol (approximately 162 fmole/mg DNA); b) a second with a lower affinity (Kd of 30–40 nM), which shows a higher capacity (approximately 860 fmole/mg DNA), and displays a saturation curve that is sigmoidal and that appears to be similar to those for Type II estrogen-binding sites in rat uterus. These results suggest that the actions of estradiol in the prostate are mediated by specific nuclear binding sites.     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.     

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites of 17β-estradiol

The interactions in vitro between [³H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing ³H-labeled steroid-protein complexes or with serum protein-bound [³H]estradiol. Nuclei preferentially accumulated ³H-labelea steroids from liver cytosol protein-steroid complexes relative to [³H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[³H]estradiol and the ³H-labeled water-soluble metabolite were retained in vitro by nuclei.     

Inactivation of thymidylate synthetase by a novel mechanism-based enzyme inhibitor: 1-(beta-D-2'-deoxyribofuranosyl) 8-azapurin-2-one 5'-monophosphate

The possible relationship of the soluble, “cytosolic” estradiol receptor with complex membranous and cytoskeletal structures of the cell matrix has been studied using a model erythrocyte system. Extraction of erythrocyte ghosts with a nonionic detergent (Triton X-100) under conditions that yield a cytoskeletal matrix reveals the presence of a limited number (less than 100) of specific sites having high affinity (k_d 10^?9 M) for the estradiol-receptor complex. The interation between the estradiol receptor and the cytoskeleton is critically dependent on temperature and it is improved by 25 mM KCl or NaCl and by 2.5 mM MgCl₂. The data suggest that the estradiol receptor, which has been generally considered to be freely “soluble” in the cytoplasm, may actually be physiologically associated in an integral manner with a complex cytoskeletal network in the cell cytoplasm.     

In vitro effects of estrogens on rat prostatic 5 alpha-reduction of testosterone

The inhibitory effects of a variety of estrogens on rat prostate testosterone Δ4–5α-reductase activity were measured by a specific $\text{in vitro}$ assay. The conversion of ³H-testosterone (initial concentration 2.8 × 10^?9 M) to labelled 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol was used as a measure of Δ4?5a-reductase activity. At a concentration of 1.8 × 10^?6 M, estradiol was the most potent inhibitor (83.4%) of the estrogens tested. Various ester derivatives, e.g. 3-acetate, 3-phosphate, were effective inhibitors. The 17-glucuronide and 3-sulfate conjugates were less effective inhibitors. The estriol isomers exerted similar degrees of inhibition (40–60%). The 3-methoxy derivatives of estradiol and estriol were poor inhibitors. The introduction of certain groups into the steroid structure, e.g. 15α-hydroxy and 6-ketone, greatly decreased the inhibitory effect of estradiol. The nature of the oxygen function at carbon 17 did not greatly influence the inhibitory effects.     

Properties of estradiol binding components in hamster uterine nuclei

Only one estrogen-binding component (Type I) was observed in salt (0.5 M KCl) extracts of proestrous hamster uterine nuclei. In addition to the classical estrogen receptor (Type I), a second binding component (Type II) was detected by [³H]estradiol exchange assay performed with hamster uterine nuclear suspensions. Although this Type II binder was not detected in salt extracts, a similar binding component was found in the nuclear debris remaining after salt extraction. The Type II binding component in the nuclear debris did not posess estrogen-binding specificity. Lack of specificity for estrogens, resistance to KCl extraction, and high capacity differentiated this Type II binder from the classical estrogen receptor. Preparation of nuclear fractions in buffer containing glycerol and monothioglycerol resulted in greater recovery of nuclear estrogen receptor (Type I) as compared to buffer lacking these constituents.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Alcohol-induced changes in hepatic estrogen-binding proteins: A mechanism explaining feminization in alcoholics

Chronic alcoholic men frequently display an apparent hyperestrogenization manifested by enhanced hepatic synthesis of estrogen-responsive proteins as well as many other estrogen-linked tissue alterations. Because of these clinical observations, we assessed the effect of chronic alcohol ingestion, using a rat model, on the levels of two estrogen-binding proteins of male rat liver cytosol. These two estrogen-binding proteins, the estrogen receptor, and an unusual male-specific estrogen binder, differ in specificity for the non-steroidal estrogen diethylstilbestrol (DES), permitting development of an assay for each using unfractionated cytosol. The estrogen receptor was labeled with [³H]DES, and the male-specific estrogen binder with [³H]estradiol in the presence of unlabeled DES, since the latter protein does not recognize DES. The specificity of labeling under these conditions was verified by gel filtration chromatography. The livers of rats fed either an alcohol-containing (AF) or isocalorically matched control diet were assayed for the levels of both proteins. The livers of the AF animals had twice the content of estrogen receptor, as compared to the isocalorically matched control group (105 vs 52 fmol/mg cytosol protein). Conversely, the livers of the AF animals had only one-third as much male-specific estrogen binder as did those of the isocalorically matched control group (22 vs 62 pmol/mg cytosol protein). Alcohol feeding also resulted in those animals having smaller testes, seminal vesicles, and prostates, as well as decreased serum testosterone levels. No change in serum estradiol levels occurred after 34 days of alcohol feeding; however, 61 days of alcohol feeding resulted in an increase in serum estradiol levels in the AF animals. These results are incorporated into a proposed model of feminization of the chronic alcoholic male.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Transformation of the rat uterine estrogen receptor after partial purification

Warming crude rat uterine cytosol after the addition of [³H] estradiol accelerates the association of the 4-S estrogen-binding protein with a second macromolecule, resulting in the formation of the 5-S estrogen-binding protein. To determine whether the 5-S estrogen-binding protein consists of two similar or dissimilar subunits, uterine cytosol was subjected to a number of fractionation procedures that separate macromolecules by solubility, molecular gel sieving, sedimentation rate, ionic charge, and heat lability. Following each of these methods, the fraction containing the 4-S estrogen-binding protein was incubated at 28°C; each of these 4-S estrogen-binding protein-containing fractions retained its capacity to completely transform to the 5-S estrogen-binding protein. In samples subjected to partial purification procedures, it was necessary that the buffer contain 40 mM Tris, 60 mM KCI, 1–10 mM dithiothreitol, and 1 M urea at pH 7.4, in order to accomplish the 4-S to 5-S estrogen-binding protein transformation at 28°C. Formation of the 5-S estrogen-binding protein requires association of the 4-S estrogen-binding protein with a molecule identical to or very similar to itself.     

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

Small nuclear RNA and VA RNA in nuclear ribonucleoprotein fibrils from adenovirus-2 infected HeLa cells

The small RNA of hnRNP¹ were studied in HeLa cells infected with adenovirus-2. At 15 h post-infection, when 50–60 % of the hnRNA was of viral origin, all the small nuclear RNA of hnRNP from non-infected cells were present in hnRNP from infected cells. The small, virus-encoded VA RNA could not be detected by staining like the snRNA but only after labeling. It represented less than 1 % of the small nuclear RNA in hnRNP. The low level of VA RNA in hnRNP as compared to that of the small nuclear RNA does not favor the hypothesis of a similar function for these 2 classes of small RNA.