Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio.

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.

ERbeta scientific visions translate to clinical uses.

Estrogen receptor beta (ERbeta) was discovered in 1995 and reported on in 1996. During the 10 years following this event, our understanding of estrogen signaling has changed remarkably. We now know that estradiol, the major endogenous activator of ER, is non-selective for the two receptors, and that ERalpha and ERbeta are, in many contexts, antagonistic against one another, an example of a yin/yang relationship, perhaps nature's way to accomplish subtle regulatory changes of estrogen signaling as a response to ever-shifting physiological requirements. Needless to say, this knowledge is of paramount significance pharmaceutically, and several ERbeta-selective agonists, intended for use against a multitude of diseases, have already been synthesized and patented by drug companies. Clearly, the next 5-10 years will be extremely exciting in view of results from clinical trials testing the clinical utility of ERbeta targeted drugs.

Maternal high n-6 polyunsaturated fatty acid intake during pregnancy increases voluntary alcohol intake and hypothalamic estrogen receptor alpha and beta levels among female offspring.

Identification of nongenetic biological factors that predispose to alcohol abuse is central to attempts to prevent alcoholism. Since an exposure to estradiol in utero increases voluntary alcohol intake in adulthood, we investigated whether an increase in pregnancy estradiol levels, caused by feeding pregnant mice a high-fat corn oil diet, also influences voluntary alcohol intake among female offspring. In addition, the effect on estrogen receptor alpha (ER-alpha) and ER-beta protein levels in the brain using Western blot assay, was determined. Pregnant CD-1 mice were kept on a high n-6 polyunsaturated fatty acid (PUFA; 43% calories from corn oil) or low n-6 PUFA diet (16% calories from corn oil) throughout gestation, and switched to a Purina laboratory chow after the pups were born. When 4 months of age, the female offspring were given a choice between 5% alcohol and tap water. The offspring of high n-6 PUFA mothers voluntarily consumed more alcohol than the offspring of low n-6 PUFA mothers. ER-alpha and ER-beta protein levels in the hypothalamus were 1.5- and 2-fold higher, respectively, in the female offspring of high n-6 PUFA mothers than in the low n-6 PUFA offspring. No significant changes in the protein levels of ER-alpha and ER-beta were seen in the frontal brain. Our findings indicate that a maternal exposure to a high n-6 PUFA diet during pregnancy increases alcohol intake among female offspring. This behavioral change, together with previously observed increase in aggressiveness and reduction in depressive-like behavior in these offspring, may be linked to an increase in the hypothalamic ER-alpha and ER-beta levels.

Cross-talk between fatty acid and cholesterol metabolism mediated by liver X receptor-alpha.

LXR alpha (liver X receptor, also called RLD-1) is a nuclear receptor, highly expressed in tissues that play a role in lipid homeostasis. In this report we show that fatty acids are positive regulators of LXR alpha gene expression and we investigate the molecular mechanisms underlying this regulation. In cultured rat hepatoma and primary hepatocyte cells, fatty acids and the sulfur-substituted fatty acid analog, tetradecylthioacetic acid, robustly induce LXR alpha (up to 3.5- and 7-fold, respectively) but not LXR beta (also called OR-1) mRNA steady state levels, with unsaturated fatty acids being more effective than saturated fatty acids. RNA stability and nuclear run-on studies demonstrate that changes in the transcription rate of the LXR alpha gene account for the major part of the induction of LXR alpha mRNA levels. A similar induction of protein level was also seen after treatment of primary hepatocytes with the same fatty acids. Consistent with such a transcriptional effect, transient transfection studies with a luciferase reporter gene, driven by 1.5 kb of the 5'-flanking region of the mouse (m)LXR alpha gene, show a peroxisome proliferator-activated receptor-alpha-dependent increase in luciferase activity upon treatment with tetradecylthioacetic acid and the synthetic peroxisome proliferator-activated receptor-alpha activator, Wy 14.643, suggesting that the mLXR alpha 5'-flanking region contains the necessary sequence elements for fatty acid responsiveness. In addition, in vivo LXR alpha expression was induced by fatty acids, consistent with the in vitro cell culture data. These observations demonstrate that LXR alpha expression is controlled by fatty acid signaling pathways and suggest an important cross-talk between fatty acid and cholesterol regulation of lipid metabolism.

Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes.

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

Interaction of the ligand-activated glucocorticoid receptor with the 14-3-3 eta protein.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

Repeated measurements of mood during psychologic treatment of dental fear.

The aims of the present study were to analyze mood changes during psychologic treatment of dental fear by assessing the rate of improvement. Twenty-one patients who refused conventional dental treatment and reported extreme dental anxiety participated in the study. Levels of dental anxiety and mood were measured with the Dental Anxiety Scale (DAS) and a Mood Adjective Checklist (MACL). MACL included two dimensions, degree of relaxation (r) and pleasantness (h) as experienced in a dental situation. Mood was monitored at each treatment session from base line to termination of the therapy (eight measurements). Two different treatment modalities were used, one with a more cognitive approach (n = 9) and one emphasizing the relaxation component (n = 12). A hierarchical linear models approach was applied to analyze individual change with repeated measurements. The results showed that positive mood changes over time were statistically significant. The mean improvement in mood scores per week and session was estimated for MACL(r) and MACL(h) to be 0.14/week and 0.09/week, respectively. The growth was not affected by DAS levels or treatment mode. This study also illustrated a powerful method for analyzing a longitudinal clinical trial design with repeated measurements.

Localization of the Rev-ErbA orphan receptors in the brain.

In the present study, we report the localization of the Rev-ErbA alpha and beta nuclear orphan receptors, two closely related members of the nuclear hormone receptor superfamily, in the brain. Both Rev-ErbA variant mRNAs were highly expressed in the olfactory bulb, the hippocampus, and in the granular cells of the cerebellum, areas enriched also in other nuclear orphan receptors. Furthermore, the alpha-isoform was found in high amounts in the frontal cortex, the superficial gray layer of the superior colliculus, and the stria terminalis. Lower expression was observed in the nucleus accumbens, the caudate-putamen, and in some thalamic and brainstem nuclei. The beta-variant, in contrast, was only moderately expressed in the cortex, mainly in the striate and retrosplenial cortices. In addition, moderate levels of Rev-ErbA beta mRNA were seen in various thalamic, pontine and brainstem nuclei. We conclude that the two Rev-ErbA isoforms share a partly similar pattern of expression in the brain, especially in areas that also contain other nuclear orphan receptors and that otherwise the localization of the two receptor subtypes is differential.

Cloning of a novel receptor expressed in rat prostate and ovary.

We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Zinc ions antagonize the inhibitory effect of aurothiomalate on glucocorticoid receptor function at physiological concentrations.

The water-soluble gold preparation aurothiomalate, which contains gold as Au(I), is frequently prescribed for patients with rheumatoid arthritis as a disease-modifying agent. We report that aurothiomalate negatively modulates glucocorticoid hormone action; it represses the ligand- and DNA-binding activities and the transactivation function of the glucocorticoid receptor. We suggested the existence of endogenous titrating activities of Au(I) because otherwise administration of aurothiomalate to a patient with rheumatoid arthritis would be expected to result in peripheral insensitivity to glucocorticoids and worsen the patient's status. Focusing on metal ions that are present in vivo, we found that Zn(II) counteracts the inhibitory effect of Au(I) on glucocorticoid receptor function. This complementary effect of Zn(II) was observed at physiological concentrations. We suggest that Zn(II) preserves glucocorticoid receptor function in target tissues and maintains hormone responsiveness, even with chrysotherapy.

OR-1, a member of the nuclear receptor superfamily that interacts with the 9-cis-retinoic acid receptor.

We have cloned a member of the nuclear receptor superfamily. The cDNA was isolated from a rat liver library and encodes a protein of 446 aa with a predicted mass of 50 kDa. This clone (OR-1) shows no striking homology to any known member of the steroid/thyroid hormone receptor superfamily. The most related receptor is the ecdysone receptor and the highest homologies represent < 10% in the amino-terminal domain, between 15-37% in the carboxyl-terminal domain and 50-62% in the DNA binding domain. The expression of OR-1 appears to be widespread in both fetal and adult rat tissues. Potential DNA response elements composed of a direct repeat of the hexameric motif AGGTCA spaced by 0-6 nt were tested in gel shift experiments. OR-1 was shown to interact with the 9-cis-retinoic acid receptor (retinoid X receptor, RXR) and the OR-1/RXR complex to bind to a direct repeat spaced by 4 nt (DR4). In transfection experiments, OR-1 appears to activate RXR-mediated function through the DR4. Therefore OR-1 might modulate 9-cis-retinoic acid signaling by interacting with RXR.

Ectopic pituitary grafts modify the response of male rats to sex differentiated promotion of diethylnitrosamine-initiated hepatic lesions with a choline-deficient diet.

The influence of implantation of ectopic pituitary grafts (PGs) under the kidney capsule in male rats on the sex differences in response to promotion with a choline-deficient (CD) diet was studied in the livers of diethylnitrosamine (DEN)-initiated Wistar rats. Growth of enzyme-altered foci, liver regeneration in response to partial hepatectomy (PH) and hepatic c-myc expression were studied. The area per enzyme-altered focus was significantly larger in initiated males fed a CD diet for 10 weeks when compared with the corresponding females. The sex difference was more pronounced 1 week after a PH performed following 10 weeks on the diet. In males carrying PGs the area per focus was reduced to the same size as in females. Liver weight gain after PH was reduced in males, but not in females, by the CD diet, and the level in PG-bearing males was intermediary, significantly different from that of males without grafts. A significantly lower labeling index in surrounding, but not in focal, hepatocytes was observed in initiated, CD-treated males than in the corresponding females 1 week after PH. In initiated as well as in uninitiated males on a CD diet the expression of the c-myc gene was 3- to 4-fold higher when compared with males fed a choline-supplemented diet at the time of PH. The mRNA level in females fed a CD diet was approximately 2.5-fold lower than in males, but still significantly above the level in females without the dietary treatment. A significant decrease in male c-myc expression was observed as a result of implantation of ectopic pituitaries. In conclusion, sex-differentiated promotion of DEN-initiated lesions with a CD diet is regulated by a pituitary influence on rat liver, in analogy with results previously obtained in the resistant hepatocyte model and with dietary deoxycholic acid promotion. This might suggest that pituitary factors are major determinants of sex-differentiated promotion in rat liver.

Adrenalectomy increases the number of substance P and somatostatin immunoreactive nerve cells in the rat lumbar dorsal root ganglia.

Using an immunocytochemical technique we have analyzed changes in substance P, somatostatin, calcitonin gene-related peptide, and galanin immunoreactivity pattern in the rat dorsal root ganglia. After 7 days of adrenalectomy, sham operated rats were compared with adrenalectomized animals either receiving a daily intraperitoneal injection of 10 mg/kg b.wt. corticosterone or vehicle. Three lumbar ganglia from each animal were blocked, serially cut, and immunostained for each neuropeptide by means of the biotin-avidin-peroxidase technique. A systematic sampling of immunoreactive ganglion cells was performed and the sample number of immunoreactive ganglion cells was calculated. After adrenalectomy, the number of substance P and somatostatin immunoreactive ganglion cells markedly increased ((means +/- S.E.M.): 245 +/- 68 versus 123 +/- 12 for sham operated animals, P < 0.01 (substance P) and 42 +/- 8 as compared to 22 +/- 9 for sham operated animals, P < 0.01 (somatostatin)). No significant changes were found in the number of calcitonin gene-related peptide and galanin immunoreactive cells after adrenalectomy. These results suggest that adrenal steroid hormones may reduce the synthesis of both substance P and somatostatin in the dorsal root ganglion cells. Daily treatment with a high dose of corticosterone, mimicking its serum levels after stress, failed to prevent the increase of peptide contents after adrenalectomy. These observations also indicate that a tonic action of corticosterone on mineralocorticoid receptors may be crucial for peptide regulation in the spinal ganglia. These results may be of relevance to adrenalectomy induced changes in sensory mechanisms, neurogenic inflammation and pain transmission and to a role of substance P and somatostatin in these processes.

Regulation of cytochrome P450 in the central nervous system.

The role of brain P450 in the physiology, pharmacology and toxicology of the brain is the subject of this study. Cytochrome P450 was isolated from the brains of rats and quantitated spectrally. The contribution of the known hepatic forms of the enzyme to the forms constitutive in the brain as well as those which are induced by hormones are xenobiotics were characterized on Western blots. We have found that the level of P450 in the brain is increased during pregnancy and lactation, by partial hepatectomy and by ethanol. In each case the profile of P450s induced is different. In pregnancy and lactation the P450 content of the hypothalamic preoptic area and olfactory lobes were increased up to 10-fold and the only subfamily identified on Western blots was 4A. There was no detectable 1A, 2A, 2B, 2C, or 2E1. Ethanol increases the level of brain P450 3- to 5-fold and P450 2C, 2E1 and 4A are induced. Upon partial hepatectomy P450 1A, 2C and 4A were detected on Western blots but there was no 2E1. The inducibility of these forms of P450 in the brain suggests that there is in situ metabolism of steroids, fatty acids, prostaglandins, ethanol and other xenobiotics in the brain and raises questions about the role of brain P450 in the development of tolerance to drugs and the neurotoxicity of xenobiotics. More importantly, the action of neurotransmitters such as dopamine which utilize fatty acids metabolites as intracellular mediators, could be influenced by the levels of 2C and 4A P450s.

Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor.

The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXR alpha cDNA was cloned and it was found that addition of in vitro-translated RXR alpha to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXR alpha, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression of RXR alpha in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.

Lactobacilli, anticarcinogenic activities and human intestinal microflora.

Lactobacilli belong to the normal oropharyngeal and intestinal microflora in humans. These microorganisms contribute to the stabilization of the microflora and maintain the colonization resistance against pathogens. Lactobacilli have been used as dietary supplements in order to prevent gastrointestinal disturbances. Claims have been made that certain strains of lactobacilli possibly exert anticarcinogenic activities. The activity of bacterial enzymes, implicated in colon carcinogenesis may be elevated by a high meat, Western-type diet. Supplements of Lactobacillus acidophilus decreased these levels in both rats and humans. Colon cancer patients given L. acidophilus fermented milk showed a significant increase both in numbers of intestinal lactobacilli and dietary calcium intake, while decreasing trends in levels of both soluble faecal bile acids and faecal bacterial enzymes, two risk makers for colon cancer, were observed. In vitro studies have revealed that lactobacilli and other lactic acid bacteria have the ability to absorb cooked food mutagens. Recent studies in humans have shown that intake of L. acidophilus significantly reduced the mutagen excretion after consumption of fried meat. Several mechanisms by which lactobacilli might exert anticarcinogenic effects are discussed. Thus, certain strains of lactobacilli might lower the colon cancer risk in humans.

Trace element status in healthy subjects switching from a mixed to a lactovegetarian diet for 12 mo.

The consequences of a change from a mixed to a lactovegetarian diet for 12 mo on trace element concentrations in plasma, hair, urine, and feces were studied in 16 women and 4 men. After the diet shift, intakes of zinc and magnesium did not change but that of selenium decreased by 40%. Three months after the diet shift, plasma and hair concentrations of zinc, copper, and selenium had decreased but those of magnesium had increased and the concentrations of mercury, lead, and cadmium in hair were lower. Also, the excretion of zinc, copper, and magnesium in urine, and that of selenium in urine and feces had decreased. Only small changes occurred during the remaining lactovegetarian-diet period. Three years later trace element concentrations had reverted towards baseline concentrations; copper values were similar to baseline concentrations but data for magnesium were slightly higher, and more complex patterns were observed for zinc and selenium. It is concluded that a shift to a lactovegetarian diet changes trace element status.