Triphenyltin isoselenocyanate: a novel nuclear retinoid X receptor ligand with antiproliferative and cytotoxic properties in cell lines derived from human breast cancer.

Several commercially available triorganotin compounds were previously found to function as agonist ligands for nuclear retinoid X receptor (RXR) molecules. Triphenyltin isoselenocyanate (TPT-NCSe), a novel selenium atom containing a derivative of triorganotin origin, was found to represent a new cognate bioactive ligand for RXRs. TPT-NCSe displayed a concentration- and time-dependent decrease in the cell viability in both human breast carcinoma MCF-7 (estrogen receptor positive) and MDA­MB­231 (triple negative) cell lines. Reactive oxygen species levels generated in response to TPT-NCSe were significantly higher in both carcinoma cell lines treated with TPT-NCSe when compared to mock-treated samples. Treatment with 500 nM TPT-NCSe caused a decrease in SOD1 and increased SOD2 mRNA in MCF-7 cells. The levels of SOD2 mRNA were more increased following the treatment with TPT-NCSe along with 1 µM all-trans retinoic acid (AtRA) in MCF-7 cells. An increased superoxide dismutase SOD1 and SOD2 mRNA levels were also detected in combination treatment of 500 nM TPT-NCSe and 1 µM AtRA in TPT-NCSe-treated MDA-MB-231 cells. The data have also shown that TPT-NCSe induces apoptosis via a caspase cascade triggered by the mitochondrial apoptotic pathway. TPT-NCSe modulates the expression levels of apoptosis­related proteins, Annexin A5, Bcl­2 and BAX family proteins, and finally, it enhances the expression levels of its cognate nuclear receptor subtypes RXRalpha and RXRbeta.

Expression of selected nuclear receptors in human epithelial ovarian cell line Caov3 exposed to bisphenol derivatives.

Objectives. Bisphenol A (BPA) is an indispensable industrial chemical. However, as a proven endocrine disruptor, it may be associated with several health disturbances, including the reproductive functions impairment and cancer. Due to the restriction of BPA usage, many bisphenol derivatives gradually substitute BPA. However, studies have reported adverse biological effects of BPA analogs, but the specific sites of their action remain largely unknown. Nuclear receptors (NRs) appear to play significant roles in various types of cancer. In addition, they are considered relevant targets of bisphenols. In the present study, we investigated the effects of BPA and its analogs bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF) on mRNA expression of selected NRs in the human ovarian epithelial cell line Caov3. The NRs examined included retinoic acid receptor α (RARA), retinoid X receptor α (RXRA), peroxisome proliferator activating receptor ß/δ (PPARD), chicken ovalbumin upstream promoter-transcription factor 2 (COUPTFII), and nuclear receptor-related protein 1 (NURR1). Methods. Caov3 cells were treated with the bisphenols at the concentrations of 1 nM, 100 nM, 10 µM and 100 µM. After 24 h and 72 h of incubation, cell viability was determined by the MTS assay, and the selected genes expression was analyzed using RT-qPCR. Results. Bisphenol treatment did not affect Caov3 cell viability, except the significant impairment after exposure to the highest BPAF dose (100 µM). At lower doses, neither bisphenol analog altered the expression of the NRs. However, at the highest concentration (100 µM), BPAF and BPA altered the mRNA levels of PPARD, COUPTFII, and NURR1 in a time- and receptor-specific manner. Conclusions. The effects of bisphenols on the specific NRs in the epithelial ovarian cancer cells were addressed for the first time by the present study. Although generally we did not find that bisphenols may provoke significant alterations in the expression of the selected NRs in Caov3 cells, they may alter mRNA expression of certain NRs at high concentrations.

The effect of all-trans retinoic acid on the mitochondrial function and survival of cardiomyoblasts exposed to local photodamage.

Using H9C2 cardiomyoblasts, we have shown that all-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A, affects mitochondrial dynamics and functions. The low dose (10 nM) ATRA stimulates the expression of nuclear retinoid receptors and induces mechanisms that are protective against severe local damage caused by laser irradiation at the mitochondrial level. These changes include increased density of the mitochondrial network, higher number of mitochondrial junctions, and enhanced mitochondrial velocity. Moreover, the treated cells had lower basal level of reactive oxygen species (ROS) and could maintain mitochondrial potential (ΔΨm ) after photodamage. Cells treated with 10 nM ATRA had significantly better survival rate after photodamage in comparison to control cells. Cells treated with pharmacological concentration of ATRA (1 µM) expressed higher mitochondrial connectivity without increased motility, which did not lead to better survival or decreased ROS level as was in the case of low-dose ATRA. The proteomics analysis showed changes in proteins related to cellular metabolism (glycolysis) and respiration in ATRA-treated cells. The l-lactate assay confirmed the shift to anaerobic glycolysis in cells treated with 1 µm ATRA, whereas the 10 nM ATRA decreased the level of lactate in medium. The increased levels of cytochrome c or peroxiredoxins 5 level and also lower expression of retinoid and rexinoid receptors were observed in cells treated with 1 µM ATRA. The effect of ATRA is concentration-dependent; the increased mitochondrial dynamics and slower metabolism at 10 nM ATRA contributed significantly to the chance of survival of the cells after photodamage whereas the higher concentration of ATRA overrode the protective effect and led to the unfavorable ones.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via ß-adrenergic signaling. However, the potential of ß-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the ß-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

CD44 and vimentin, markers involved with epithelial-mesenchymal transition: A proteomic analysis of sequential proteins extraction of triple-negative breast cancer cells after treatment with all-trans retinoic acid.

This work aimed to provide, in one isolation and separation step, an overview of the content of proteins with different solubility after treatment with all-trans retinoic acid, which is considered to be an important therapeutic agent, predominantly in acute promyelocytic leukemia. Breast, ovarian, bladder, and skin cancers have been demonstrated to be suppressed by retinoic acid, as well. The bottom-up proteomic strategies were applied for the analysis of proteins extracted from triple-negative breast cancer MDA-MB-231 cells utilizing a commercially manufactured kit. The gel electrophoresis followed by MALDI-TOF MS analysis was used for protein determination. By employing PDQuest™ software, we identified several proteins affected by all-trans retinoic acid. Two proteins, vimentin and CD44, which are associated with the epithelial-mesenchymal transition, were selected for a detailed study. We have found that all-trans retinoic acid results in significantly reduced levels of vimentin and CD44 in both the cytoplasmic and membrane fractions. A significant effect was particularly evident in CD44, where protein level in the cytoplasmic fraction was almost completely suppressed.

Novel insights into the combined effect of triorganotin compounds and all-trans retinoic acid on expression of selected proteins associated with tumor progression in breast cancer cell line MDA-MB-231: Proteomic approach.

Trialkyltins and triaryltins function as nuclear retinoid X receptors (RXR) agonists due to their affinity to the ligand-binding domain of RXR subtypes and function as transcriptional activators. We present the data on combined effects of all-trans retinoic acid (ATRA), retinoic acid receptor (RAR) ligand and tributyltin chloride or triphenyltin chloride (RXR ligands) on protein pattern in MDA-MB-231 cells. Proteomic strategies based on bottom-up method were applied in this study. The total cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. By employing PDQuest™ software, we identified more than 30 proteins differently affected by the above compounds. For further studies, we selected specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or to cellular processes as apoptosis, regulation of gene transcription or epithelial-mesenchymal transition (annexin 5, nucleoside diphosphate kinase B and vimentin). We have found that treatment of MDA-MB-231 cells with triorganotins reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin and nucleoside diphosphate kinase B. These results demonstrate that RXR/RAR heterodimer may act under this experimental design as permissive heterodimer allowing activation of RXR by triorganotins.

Effects of natural ligands and synthetic triorganotin compounds of nuclear retinoid X receptors in human MCF-7 breast cancer cell line.

In the present study, we analyzed in vitro effects of natural and synthetic triorganotin ligands of nuclear retinoid X receptors in human MCF-7 breast cancer cells. Our data has shown that all-trans retinoic acid significantly reduced expression of RXRalpha mRNA, Bcl2 and enhanced expression of BAX proteins. Tributyltin bromide markedly decreased mRNA level of RXRalpha and RXRbeta. Significantly reduced levels of both RXRs proteins were observed after treatment with tributyltin chloride (TBT-Cl) but not after treatment with triphenyltin chloride (TPT-Cl) for RXRbeta protein. Both RXRalpha and RXRbeta protein levels decrease was found also by combination ATRA+TBT-Cl/TPT-Cl.

A comparative study of protein patterns of human estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines.

In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently.

Agonistic effect of selected isoflavones on arylhydrocarbon receptor in a novel AZ-AhR transgenic gene reporter human cell line.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. Structurally diverse compounds bind to AhR and act as AhR agonists. Well characterised family of natural AhR ligands are isoflavones, which are compounds found predominantly in soy beans or red clover. In this study we have examined agonistic effect of selected isoflavones (genistein, daidzein, biochanin A, formononetin and equol) on AhR in the novel transgenic gene reporter human cell line AZ-AhR, a stably transfected AhR-responsive cell line allowing rapid and sensitive assessment of AhR transcriptional activity. We demonstrated that biochanin A, formononetin and genistein at concentration 10(-4) mol/l exerted agonistic effects on AhR with fold activation of 309- fold, 108-fold and 27-fold, which is about 84.8%, 29.6% and 7.4%, respectively, of the value attained by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Daidzein and equol did not show any significant effects on AhR.

Nuclear receptors - target molecules for isoflavones in cancer chemoprevention.

Breast cancer is the most occurring type of cancer among women. In Slovakia, there are yearly diagnosed about 1900 new cases of this disease. Breast cancer treatment is very expensive, psychic stressful and in some cases ineffective. Therefore, it is essential to search for new and/or alternative methods for breast cancer treatment, since nuclear receptors are considered to be a central goal for maximizing treatment opportunities in breast cancer. Among natural ligands for estrogen receptors (ERα and ERß), which are member of nuclear receptors superfamily, belongs also isoflavones. These natural compounds have similar structure to main female hormon-17ß estradiol. A rich source of isoflavones is soy and its products. Three aglycones form of isoflavones (genistein, daidzein, glycitein) are predominantly found in soybean and red clover. Among other important isoflavones belongs also biochanin A and formononetin.

Changes in retinoic acid receptor status, 5'-deiodinase activity and neuroendocrine response to voluntary wheel running.

Little information is available on the involvement of retinoic acid in processes related to physical activity. The aim of this study was to test the hypotheses that long-term voluntary wheel running (1) modifies RARs concentration as well as the expression of RAR subtypes and (2) alters Iiodothyronine deiodinase (5'-DI) activity in rat liver. To evaluate relevant mechanisms, hepatic gene expression of specific nuclear receptor coregulators and stress hormone levels in plasma have also been measured. Sprague-Dawley rats were housed either in standard cages or in cages with access to running wheel attached for 3 weeks. RAR maximal binding capacity in the liver was found to be significantly lower while gene expression of RAR beta increased in rats exposed to voluntary running compared to that in sedentary controls. Gene expression of RAR alpha, RXR alpha and RXR beta was found to be unaffected. Voluntary running led to a significant decrease of 5'-DI activity in the liver. No significant changes in the gene expression of specific nuclear receptor coregulators in the liver were observed. Significant elevation of aldosterone while no changes in ACTH and corticosterone concentrations were observed in rats exposed to wheel running compared to those in controls. In conclusion, this study provided first evidence on the reduction of liver RAR concentrations and 5'-DI activity in response to long-term voluntary wheel running. Neuroendocrine mechanisms involved in these changes may include adrenal mineralo- and glucocorticoids.

Down-regulation of vimentin by triorganotin isothiocyanates-nuclear retinoid X receptor agonists: A proteomic approach.

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.

Different mRNA expression profiling of nuclear retinoid, thyroid, estrogen and PPARgamma receptors, their coregulators and selected genes in rat liver and spleen in response to short-term in vivo administration of 13-cis retinoic acid.

Retinoic acids (RAs) and also their analogs (synthetic retinoids and rexinoids) have been regarded as major therapeutic and/or chemopreventive agents and can regulate a number of diverse processes-such as immune system, hormonal systems. In this work we describe different effects of short-term treatment of Wistar male rats with 13-cis retinoic acid on the regulation of retinoic acid receptors (RARs), retinoid-X receptors (RXRs), thyroid hormone receptors (TRs), ERs, 5'-DI, EGFR and erb-B2/neu genes in liver and/or spleen. Using RT-PCR analysis we have found that administration of 13-cis retinoic acid enhanced expression of RARbeta and PPARgamma mRNA, and decreased expression of RARalpha, RARgamma, RXRbeta and TRbeta mRNA in liver. On the other hand, in spleen this treatment resulted in decreased expression of RARalpha, RARbeta, RARgamma, TRalpha and ERbeta mRNA. Our findings indicate distinct modulation of various signal pathways by short-term administration of 13cRA, which also differ in spleen when compared to liver. We suggest that even a short-term treatment of rats with 13cRA may affect a reasonable number of steps in retinoid signaling pathways, a number of which might be very likely extended by long-term treatment of mammals by 13-cis retinoic acid.

Vitamin D3 affects expression of thyroid hormone receptor alpha and deiodinase activity in liver of MNU-treated Sprague-Dawley rats.

1alpha,25-dihydroxyvitamin D3 and its analogue, Seocalcitol (EB1089), are able to reverse or slow the process of carcinogenesis in experimental models and cell cultures. The aim of this study was to investigate the effect of administration vitamin D or Seocalcitol to female Sprague-Dawley rats with 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis of mammary glands on binding characteristics and mRNA levels of thyroid hormone receptors (TRs). Chemopreventive administration of vitamin D caused significant reduction of animal body weight. The expression of TRalpha mRNA was significantly higher in liver of animals treated with vitamin D after detection of first tumour. In our experiment, administration of vitamin D or Seocalcitol significantly reduced KA (group MNU+Seo; MNU+D) and increased Bmax (group MNU+Seo) of thyroid receptors in liver when compared to healthy animals. We show that the activity type I 5'-deiodinase was significantly decreased in livers of animals treated with vitamin D. The data from our in vivo experiment has clearly shown, for the first time, that vitamin D but not Seocalcitol i) may affect the body weight of animals, ii) can cause an increase in the expression of TRalpha in rat liver, remaining the functionality of the TRs unaffected, and iii) is responsible for type I iodothyronine 5'-deiodinase activity decrease in rat liver, remaining the expression of the enzyme unaffected.

Causal associations of autoimmune thyroiditis and papillary thyroid carcinoma: mRNA expression of selected nuclear receptors and other molecular targets.

Potential causal associations of autoimmune thyroiditis (AIT) and papillary thyroid carcinoma (PTC) have been studied previously. The mRNA expression patterns of thyroid hormone receptors (TR), retinoid receptors (RAR), rexinoid receptors (RXR), dihydroxyvitamin D3 receptors (VDR), and progesterone receptors (PR) in PTC tissue of patients without autoimmune thyroiditis (PTC/AIT-) and in PTC tissue of patients with coexisting AIT (PTC/AIT+) have been investigated in order to judge whether the observed changes may take part in the promotion and progression of thyroid cancer. Tumours with or without AIT were classified histologically and the semiquantitative PCR was performed. The results revealed that there was decreased expression of TRα, TRßα, RARα and PR mRNA in PTC/AIT+ tumours when compared with PTC/AIT- tumours. Decreased expression of RARα in PTC/AIT+ was detected when compared with PTC/AIT- patients. A similar effect of AIT was observed with a decrease in RARγ expression in PTC/AIT+ patients. On the other hand, there was an increased expression of VDR in thyroid tumours (PTC/AIT+) when compared with PTC/AIT-. PR mRNA was decreased in the thyroid tumours of PTC/AIT+ patients when compared with PTC/AIT- patients. In addition, there was an increased expression of MKi67 and complement C3 in PTC of PTC/AIT+ when compared with PTC/AIT-. In the PTC/AIT+ group, a decreased level of IGF-1 mRNA was found when compared with the PTC/AIT- group. According to the significant differences of the studied markers in PTC/AIT+ compared with PTC/AIT-, it was indicated that AIT may be a predisposing factor for the development of PTC.

Combined treatment of P-gp-positive L1210/VCR cells by verapamil and all-trans retinoic acid induces down-regulation of P-glycoprotein expression and transport activity.

The development of the most common multidrug resistance (MDR) phenotype associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells may result in more than one hundred fold higher resistance of these cells to several drugs. L1210/VCR is a P-gp-positive drug resistant cell line in which P-gp overexpression was achieved by repeated cultivation of parental cells with a stepwise increasing concentration of vincristine. Relatively little is known about regulation of P-gp expression. Therefore, serious efforts have been made to recognize all aspects involved in regulation of P-gp expression. Retinoic acid nuclear receptors are involved in regulating expression of a large number of different proteins. Several authors have described that all-trans retinoic acid (ATRA, ligand of retinoic acid receptors, RARs) may induce alterations in P-gp expression and/or activity in drug resistant malignant cell lines. There are also other nuclear receptors for retinoids--retinoid X receptors (RXRs)--that may be involved in the development of the P-gp-mediated MDR phenotype. The topic of the present paper is a study of the relationship, if any, between the regulatory pathways of nuclear receptors for retinoids and P-glycoprotein expression. Increased levels of mRNAs encoding the retinoic acid nuclear receptors RARalpha and gamma, as well as decreased levels of the mRNAs encoding RARbeta and the retinoid X receptor RXRgamma or slightly decreased levels of RXRbeta mRNA, were observed in L1210/VCR cells in comparison with parental L1210 cells. Neither L1210 cells nor L1210/VCR cells contained measurable amounts of mRNA encoding the RXRalpha receptor. ATRA did not influence the viability of L1210/VCR cells differently from L1210 cells. A combined treatment of L1210/VCR cells with vincristine (1.08 micromol/l) and ATRA induced slightly higher cell death than that observed with ATRA alone. When applied alone, ATRA did not influence P-gp expression (monitored by anti P-gp antibody c219 using western blot analysis) or transport activity (monitored by use of calcein/AM as a P-gp substrate by FACS) in L1210/VCR cells. In contrast, when ATRA was applied together with verapamil (an often used P-gp inhibitor), a significant decrease in P-gp expression and transport activity were observed. However, no significant differences in [11, 12-(3)H]-ATRA uptake were observed in either sensitive or resistant cells, in the latter case in the absence or presence of vincristine. Moreover, verapamil did not influence ATRA uptake under any conditions. Thus, we can conclude that the combined treatment of L1210/VCR cells with ATRA and verapamil is able to depress P-gp expression, and consequently its activity. ATRA is not a P-gp-transportable substance, and thus this effect could not be attributed to verapamil-induced inhibition of P-gp that would allow ATRA to reach retinoic acid nuclear receptors and activate them.

Expression, protein stability and transcriptional activity of retinoic acid receptors are affected by microtubules interfering agents and all-trans-retinoic acid in primary rat hepatocytes.

Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all-trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1microM) down-regulated RARalpha and RARgamma mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARbeta mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC

Evaluation of estrogenic potency of a standardized hops extract on mammary gland biology and on MNU-induced mammary tumor growth in rats.

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E2-benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms.

The role of retinoic acid receptors and their cognate ligands in reproduction in a context of triorganotin based endocrine disrupting chemicals.

Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.

Radioligand binding assay for accurate determination of nuclear retinoid X receptors: A case of triorganotin endocrine disrupting ligands.

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.