Biomedical subjects

P Diel

Publications and source records attributed to P Diel.

At least 19 recordsLinked to original sources

PubMed2006

17beta-hydroxy-5alpha-androst-1-en-3-one (1-testosterone) is a potent androgen with anabolic properties.

Since the begining of the year 2005, the use of steroid precursors (prohormones) is illegal in the United States; nevertheless, there is still an enormous abuse of such substances. One of the most frequently misused steroids, often declared to be a prohormone, is 1-testosterone (17beta-hydroxy-5alpha-androst-1-en-3-one, 1-Testo). In this study, we have characterised molecular mechanisms of its action, determined its tissue specific androgenic and anabolic potency and investigated potential adverse effects. 1-Testo binds highly selective to the androgen receptor (AR) and has a high potency to stimulate AR dependent transactivation. In vivo an equimolar dose of 1-Testo has the same potency to stimulate the growth of the prostate, the seminal vesicles and the androgen sensitive levator ani muscle as the reference compound testosterone propionate (TP). Administration of 1-Testo, in contrast to TP, results in a significant increase of liver weight. Our results demonstrate that 1-Testo, even without being metabolised, is a very potent androgen. It binds selectively to the AR and transactivates AR dependent reporter genes. In vivo it has a high androgenic and anabolic potency and increases liver weight. In summary 1-Testo can be characterised as a typical anabolic steroid. It has to be assumed that consumption of this substance is associated with adverse side effects typical for this class of compounds. Therefore, a strict control of its ban is essential.

Anabolic Agents↗

PubMed2006

Toxicokinetics of bisphenol A in pregnant DA/Han rats after single i.v. application.

Bisphenol A (BPA) is an important chemical in the production of epoxy resins and polycarbonate plastics, and basic monomers which are used for a variety of applications. Consumer exposure to BPA may be possible from migration of BPA from dental sealants or from polycarbonate or epoxy-lined food and drink containers. BPA is known to act as weak estrogen mimic in rodents, and there is a concern of adverse endocrine effects, especially from prenatal exposure to this potential 'endocrine disruptor'. To address this concern, we have studied the disposition and transplacental transfer of BPA in pregnant DA/Han rats on day 18 of gestation. The BPA concentrations were determined by GC/MS analysis in maternal blood, maternal organs (liver, kidney, uterus), placenta and fetuses (fetal liver and residual tissues) at different time-points (5-360 min) after intravenous administration of 10 mg BPA/kg body weight. Total BPA (aglycone and conjugates) was analyzed in all tissue samples after enzymatic hydrolysis and liquid/liquid extraction; in maternal plasma, total BPA and BPA aglycone were analyzed in parallel samples (with/without hydrolysis). Soon (5 min) after the i.v. injection a mean total BPA concentration of 3.8 microg/ml was found in maternal plasma; it declined in the first 2 h to 0.7 microg/ml. Early after injection, the majority of circulating BPA (almost 80%) was still in the aglycone form, but, metabolism by phase II enzymes decreased the BPA aglycone concentration to 0.3 microg/ml after 2 h. Despite this efficient conjugation, BPA was rapidly distributed in the organism: In well perfused organs peak concentrations for total BPA were attained 20-30 min after intravenous administration, with mean values of about 9.7 microg/g in maternal liver, 8.6 microg/g in kidneys, and 6.2 microg/g in the uterus. The peak values in other tissues were lower, with 4.0 microg/g for placenta, 3.3 microg/g for fetal liver, and 2.4 microg/g for residual fetus homogenate. The BPA levels in all tissues thereafter declined more or less in parallel with those in maternal blood. The rather similar concentration time course in placenta and fetal liver indicates that BPA is readily transferred across the placenta of DA/Han rats to the fetus. Our data on BPA disposition in DA/Han rats are discussed in the context of other kinetic studies with BPA in pregnant rats, and in relation to the previous results from our laboratory (Degen et al. Arch Toxicol 76:23-29, 2002a, b, c) demonstrating comparable transplacental transfer of daidzein, a phytoestrogen that accounts for a significant portion of total human exposure to potential endocrine disruptors.

Animals↗

PubMed2005

Tetrahydrogestrinone is a potent but unselective binding steroid and affects glucocorticoid signalling in the liver.

Tetrahydrogestrinone (THG) is a steroid recently identified to be misused as doping agent. However, the knowledge on functions of this substance in humans or animal models is rather limited. Therefore, it was our aim to further characterize the pharmacological profile of THG and identify potential adverse side effects. THG was synthesized, the purity was confirmed and its biological activity was tested. The potency of THG to transactivate AR dependent reporter gene expression was two orders of magnitude lower compared to dihydrotestosterone. THG binds with high affinity but unselective to the androgen (AR), progesterone (PR), glucocorticoid (GR) and mineralocorticoid (MR) receptor. Treatment of orchiectomised rats with THG resulted in a stimulation of prostate, seminal vesicle and levator ani muscle, indicating androgenic and anabolic properties. In the liver THG, in contrast to testosteronepropionate (TP), down regulates the expression of the GR dependent tyrosine aminotransferase gene (TAT). In summary, our results demonstrate that THG is not a specific AR agonist. THG exhibits a high binding affinity to all tested steroid hormone receptors and binds with highest affinity to the GR. Our in vivo data are indicative of an anabolic and androgenic potency of THG, but the repression of TAT demonstrates that THG also interferes with the glucocorticoid hormone system. Therefore, it is conceivable that an intake will result in adverse side effects.

Anabolic Agents↗

PubMed2005

Tissue-specific modulation of cyclooxygenase-2 (Cox-2) expression in the uterus and the v. cava by estrogens and phytoestrogens.

Cyclooxygenase 2 (Cox-2), an enzyme involved in prostaglandin production, is a key player in the development of pathologic changes, such as colorectal cancer, arteriosclerosis and thrombosis. In this study, we investigated the effects of estrogens, selective estrogen receptor modulators (SERMs), pure antiestrogens and phytoestrogens on the tissue-specific expression of Cox-2 in the uterus and the v. cava of ovariectomized female rats. Cox-2 expression could be detected in the uterine epithelium and in the endothelium of the v. cava. Cox-2 expression was time-dependently stimulated after administration of 17beta estradiol (E2) in the uterus. In the v. cava, E2 treatment resulted in a stimulated expression of the progesterone receptor (PR), a gene known to be regulated by E2, whereas Cox-2 was simultaneously down-regulated. Administration of the pure antiestrogen faslodex (Fas) had no effect on Cox-2 expression. In contrast, administration of tamoxifen (Tam) resulted in a decrease of Cox-2 expression in the v. cava but does not stimulate Cox-2 expression in the uterus. Interestingly, the same expression pattern of Cox-2 could be detected after dose-dependent administration of genistein (Gen). Here, down-regulation of Cox-2 could already be detected after administration of merely 0.5 mg/(kgBW) Gen, a dose where no effects on uterine weight were observed. In summary, our results demonstrate a reverse tissue-specific regulation of Cox-2 expression by estrogens in the v. cava and uterus indicating the existence of complex molecular mechanisms which have to be characterized in future studies. Remarkably, Tam and the phytoestrogen Gen, both share the ability to decrease the expression of Cox-2 in the v. cava without effecting its uterine expression. These observations may be of great importance with respect to potential beneficial or adverse effects of estrogens, SERMs and phytoestrogens on the cardiovascular tissue.

Animals↗

PubMed2005

The growth factor myostatin, a key regulator in skeletal muscle growth and homeostasis.

Skeletal muscle possesses the ability to both respond and adapt to changing environmental stimuli, leading to a set of metabolic and morphological adaptations, which allow it to better meet the energy demands of sustained physical activity. Great progress has been achieved over the past years by means of innovative molecular techniques, which has led to the discovery of new growth factors and the identification of molecular mechanisms involved in the regulation of muscle development. These findings provide new starting points to understand the molecular mechanisms involved in the adaptation of skeletal muscle to exercise training. One of these new identified growth factors is myostatin, a member of the transforming growth factor-beta family of proteins that has been demonstrated to play a fundamental role in the regulation of skeletal muscle growth during embryogenesis. Blocking of the myostatin signalling transduction pathway by specific inhibitors and genetic manipulations has been shown to result in a dramatic increase of skeletal muscle mass. This review focuses on the importance of myostatin in mediating skeletal muscle homeostasis in response to training as well as during the progress of myogenic disease, like atrophy or dystrophy. Manipulations of myostatin signalling may be useful for agriculture applications, treatment of muscle diseases, inhibition of muscle atrophy and last but not least as life style drugs in antiaging therapies or manipulations of the muscle to fat ratio. Drugs with the ability to modulate myostatin signalling may have the potential to enhance physical performance in athletes and therefore they probably represent a new class of doping substances.

Animals↗

PubMed2005

Short-term endurance training results in a muscle-specific decrease of myostatin mRNA content in the rat.

AIM: Myostatin has been characterized as a negative regulator of skeletal muscle growth. To examine a probable function of myostatin during the adaptation of skeletal muscle in response to training, we analysed the effect of short-term endurance training on myostatin and insulin-like growth factor-I (IGF-I) mRNA contents in rat skeletal muscles. To assess the impact of the training stimulus, mRNA levels of metabolic genes were analysed simultaneously. METHODS: Male Wistar rats were trained for 5 days by swimming, while another group remained untrained. Myostatin, IGF-I, glucose transporter 4 (GLUT4), hexokinase II (HK II) and hydroxyacyl-CoA dehydrogenase (HAD) mRNA levels were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in gastrocnemius, vastus lateralis and soleus muscles. A time course experiment was conducted, in order to examine transient changes of myostatin mRNA contents in gastrocnemius 7 and 24 h after one-swimming session as well as 24 h after a 3-day swimming training. RESULTS: No significant changes in IGF-I and GLUT4 mRNA levels were found in any of the muscles analysed. mRNA contents of myostatin were significantly reduced in gastrocnemius and vastus lateralis but not in soleus. In agreement to this pattern, we found significantly higher mRNA levels of HK II and HAD in the trained group. The time course experiment revealed significantly reduced myostatin mRNA contents in gastrocnemius 7 but not 24 h post-exercise. The 3-day swimming training resulted also in significantly lower myostatin mRNA levels in the trained group. CONCLUSION: This study demonstrated that short-term endurance training may modulate myostatin mRNA levels, implying a probable role of myostatin in remodelling of skeletal muscle in response to training.

Animals↗

PubMed2004

Effect of voluntary exercise on the expression of IGF-I and androgen receptor in three rat skeletal muscles and on serum IGF-I and testosterone levels.

The effects of anabolic agents and training on skeletal muscle are believed to be mediated by a variety of growth and transcription factors. Among these regulatory proteins, insulin-like growth factor-I (IGF-I) and androgen receptor (AR) play a crucial role. The purpose of this study was to investigate the effects of wheel running on IGF-I and AR mRNA expression in three distinct rat skeletal muscles (i.e., gastrocnemius, vastus lateralis, and soleus), as well as on the serum levels of IGF-I and testosterone. Twenty male Wistar rats were housed in cages with free access to running wheels for 12 weeks, while nine rats served as controls. Analysis of the mRNA expression of IGF-I and AR using real time RT-PCR revealed no significant differences between the trained and untrained rats in any of the muscles studied. Enzyme immunoassay showed significantly lower serum levels of IGF-I and testosterone in the trained compared to the untrained animals. These results suggest that chronic exercise in wheels does not affect IGF-I and AR mRNA levels in rat skeletal muscle, while decreasing the circulating levels of two anabolic factors, i.e., IGF-I and testosterone. It is concluded that IGF-I, AR and testosterone seem to play a marginal role during the adaptation process of rat skeletal muscle to long-term wheel running.

Animals↗

PubMed2003

Comparative responses of three rat strains (DA/Han, Sprague-Dawley and Wistar) to treatment with environmental estrogens.

The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Although much attention has been paid to identifying protocol variables and reproducibility between laboratories the question whether toxicodynamic and toxicokinetic variations of different strains may affect their sensitivity to estrogenic stimuli has been rarely addressed. We have compared the estrogenic activity of the environmental chemicals genistein (GEN), bisphenol A (BPA) and p- tert-octylphenol (OCT) in DA/Han (DA), Sprague-Dawley (SD) and Wistar (WIS) rats after repeated oral application. Rats were treated per os for 3 days with different doses of these weakly estrogenic compounds and the potent reference estrogen ethinylestradiol (EE). Then uterine wet weight, thickness of the uterine epithelium, uterine gene expression of clusterin (CLU), and thickness of the vaginal epithelium were examined as parameters for estrogenic potency of the test compounds in the three strains of rats. The uterotrophic response to treatment with BPA, OCT and GEN was similar in the three strains, and allowed us to rank them as GEN being more potent than OCT, and BPA being the weakest estrogen. This was confirmed by analysis of other biological endpoints, despite some differences in the magnitude of their response among strains and to distinct compounds. For instance, the uterus wet weight response to EE treatment indicated lower sensitivity of SD rats than that of DA and WIS rats, but this was not observed for responses of the uterine or vaginal epithelium. Moreover, blood concentrations were assessed at the time of killing and related to biological responses: plasma levels of total and unconjugated BPA and GEN depended upon the dose administered and varied to some extent within treatment groups and among the three rat strains. However, there was no good correlation in the three strains between individual compound concentrations analysed 24 h after the last dose and the uterotrophic wet weights. Summarising our results, we conclude that the sensitivity of various biological endpoints can differ slightly between strains of rats. On the other hand, our data demonstrate that the choice of the rat strain does not lead to pronounced differences in the evaluation of estrogenic activities of chemicals, especially when different biological endpoints are included in the analysis.

Administration, Oral↗

PubMed2002

In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.

Animals↗

PubMed2002

Transplacental transfer of the phytoestrogen daidzein in DA/Han rats.

Disposition and transplacental transfer of the phytoestrogen daidzein was studied in pregnant DA/Han rats on day 18 of gestation. Daidzein concentrations were determined by HPLC in maternal blood, maternal organs (liver, kidney, uterus), placenta and fetuses (liver and residual tissues) at specific times (5, 10, 20, 40 and 120 min) after intravenous administration of 10 mg/kg body weight. Early after injection, the majority of circulating daidzein was still in the aglycone form; at later time points the majority consisted of conjugates. The initially high isoflavone concentration in maternal plasma (about 25 microg/ml at 5 min) decreased rapidly within the first hour, and after 2 h total daidzein was below 1 microg/ml. Despite its efficient conjugation, daidzein was rapidly distributed in the organism: peak concentrations were attained 10 min after intravenous administration in all tissues analysed, with mean values of about 31 microg/g in maternal liver, 13 microg/g in kidneys and 5 microg/g in the uterus. Placenta contained about one-tenth the hepatic daidzein concentration, and fetal liver about 1/30 the peak concentration of maternal liver (i.e. 1.3 microg/g, which is one-third the placental concentration). Daidzein levels in tissues then declined in parallel with those in maternal blood. The data show that daidzein is transferred across the placenta of DA/Han rats to fetuses. This is indicative of a rapid transfer from the mother to the fetus, but also that efficient hepatic extraction of daidzein from the maternal blood occurs. Since dietary phytoestrogens account for a significant proportion of human exposure to potential endocrine modulators, and since the placenta does not represent a barrier to daidzein or related estrogenic isoflavones, the consequences of these exposures early in life should be examined and monitored carefully.

Animals↗

PubMed2001

Molecular identification of potential selective estrogen receptor modulator (SERM) like properties of phytoestrogens in the human breast cancer cell line MCF-7.

Numerous epidemiologic studies revealed that ethnic populations with higher dietary intake of phytoestrogens have the lowest incidence for breast cancer. The molecular mechanisms which may be responsible for this cancer protective action of phytoestrogens are so far only barely characterised. There are some hints that phytoestrogens may act like selective estrogen receptor modulators (SERMs) on the breast. For this reason we have investigated potential SERM-like properties of the phytoestrogens daidzein (Dai), coumestrol (Cou), and genistein (Gen) in the human breast cancer cell line MCF-7. Effects of these substances on progesterone (PR) and estrogen receptor alpha (ER) mRNA expression and estrogen receptor alpha protein levels were studied in comparison to estradiol (E2) and the synthetic SERMs raloxifene (Ral) and faslodex (ICI 182 780). PR mRNA expression was up-regulated after administration of Cou, whereas treatment with Dai and Gen induced only a faint increase. ER mRNA expression was down-regulated by Cou but not affected by Dai and Gen. The content of ER protein in the breast cancer cells was strongly decreased by Gen, only a faint reduction could be observed following administration of Cou, whereas administration of Dai slightly increases ER protein levels. In summary and in comparison to the effects observed after administration of E2, Ral, and ICI it turned out that Cou shows molecular properties which are very similar to an estrogen receptor agonist like E2, whereas the molecular properties of Gen are comparable to the SERMs ICI and Ral. These results clearly indicate that phytoestrogens differ significantly in regard to their molecular action on breast cancer cells and can be subdivided into distinct functional categories.

Blotting, Western↗

PubMed2001

Phytoestrogens and carcinogenesis-differential effects of genistein in experimental models of normal and malignant rat endometrium.

The phytoestrogen genistein was studied in normal and malignant experimental uterine models in vivo. The action of genistein on the uterus and vagina of ovariectomized DA/Han rats after 3 day oral administration (25, 50 or 100 mg/kg/BW/d) was compared to ethinyl oestradiol (0.1 mg/kg/BW/d). Effects on uterine and vaginal morphology, uterine growth and uterine gene expression were studied. A dose dependent increase of the uterine wet weight and the uterine and vaginal epithelial height, a dose dependent up-regulation of complement C3, down-regulation of clusterin mRNA expression and a stimulation of the vaginal cornification was observed after administration of genistein. Uterine gene expression and vaginal epithelium respond to genistein at doses where no significant effects on uterine wet weight were detectable. In general the vagina was more sensitive to genistein than the uterus. To analyse the action of genistein in malignant uterine tissue, the impact of a 28 d treatment with 50 mg/kg/d of genistein on the in-vivo tumour growth of RUCA I endometrial adenocarcinoma cells, following subcutaneous inoculation into syngeneic DA/Han rats, was assessed. In contrast to ethinyl oestradiol (0.1 mg/kg/BW/d), a dose of 50 mg/kg/BW/d of genistein did not affect tumour growth. Nevertheless C3 and TRPM2 mRNA expression in the tumour were both significantly stimulated by ethinyl oestradiol and genistein. In comparison to ovariectomized animals genistein up-regulated uterine wet weight and uterine dependent gene expression in tumour bearing animals. In conclusion, four independent uterine and vaginal parameters indicate genistein is a weak oestrogen receptor agonist in the uterus and vagina of female DA/Han rats, and evidence is provided for a selective oestrogen receptor modulator (SERM)-like action of genistein in normal and malignant uterine tissue.

Adenocarcinoma↗

PubMed2000

Toxicokinetics of bisphenol A in female DA/Han rats after a single i.v. and oral administration.

Bisphenol A [BPA; 2,2-bis-(4-hydroxyphenyl)-propane] is a monomer used in the manufacture of resins with a wide range of applications, e.g. plastic coatings in the food packaging industry. BPA has been shown to have a weak oestrogenic activity in vitro and in vivo. Despite its low oestrogenic potency there is concern that, as a consequence of slow clearance, BPA might reach biologically significant levels in humans and animals exposed to environmental levels. To address this concern, we assessed the kinetic behaviour of BPA in female DA/Han rats. Groups of female rats received 10 mg BPA/kg body weight intravenously or 10 or 100 mg BPA/kg body weight orally (by gavage). Blood samples were collected at different time-points and plasma was prepared. Free BPA in the samples was isolated by fluid-fluid extraction. BPA was measured by GC-MS which allowed the reliable determination of BPA concentrations as low as approximately 10 ng/ml plasma. Immediately after i.v. administration, the BPA plasma concentration was in the range of about 15 microg/ml and decreased rapidly within the first hour (to 700 ng/ml). The levels declined further (100 ng/ml at 2 h), and after 24 h the analytical detection limit was reached. BPA was detected in plasma as early as 10 min after gavage administration, indicating rapid initial uptake from the gastrointestinal tract. Absorption of BPA was variable. In animals receiving 10 mg/kg, maximal plasma levels were reached after 1.5 h (31 ng/ ml) and 6 h (40 ng/ml). In animals receiving 100 mg/kg, plasma levels reached maxima around 30 min (150 ng/ml) and 3 h (134 ng/ml) after administration. After 48 h BPA was at or below the detection limit in both dose groups. Fluctuations in the BPA plasma concentrations over time point to the possibility of enterohepatic recirculation and protracted absorption from the gastrointestinal tract. Using the area under the concentration-time curves (AUCs), low bioavailabilities of 16.4% and 5.6% were calculated for the 10 and 100 mg/kg dose groups, respectively. The toxicokinetic properties of BPA in DA/Han rats are in agreement with the hypothesis of a rapid first-pass elimination by the liver and efficient metabolic clearance of low oral doses. Only excessive doses may lead to bioaccumulation if detoxification pathways are saturated.

Administration, Oral↗

PubMed2000

Toxicokinetics of the phytoestrogen daidzein in female DA/Han rats.

Female DA/Han rats were given the phytoestrogen daidzein, either intravenously (10 mg/kg b.w.) or orally by gavage (10 or 100 mg/kg b.w.). The plasma concentration-time curve determined after i.v. administration of daidzein was fitted to a triexponential model, resulting in a final half-life (gamma-phase) of approximately 4 h. The oral bioavailability of 10 mg daidzein/kg was 9.7%, while that of 100 mg/kg was 2.2%; the higher dose (100 mg/kg) was apparently absorbed to a four- to fivefold lower extent than the smaller dose. The plasma concentration time curves after oral administration of daidzein to female DA/Han rats revealed pronounced interindividual differences and multiple peaks, pointing to extensive enterohepatic circulation and/or protracted absorption from the gastrointestinal tract. As shown in a separate experiment with bile duct-cannulated rats, daidzein (i.p. 10 mg/kg b.w.) is efficiently excreted with bile: glucuronide/sulfate metabolites amounting to approximately 30% of the dose in 8 h. Conjugates were also the main circulating metabolites upon i.v. or gavage administration of daidzein, indicating efficient phase II metabolism in female DA/Han rats. Since only few data have been published on tissue levels of isoflavones, their concentrations were measured in various organs and compared to plasma levels determined at the time the animals were killed, with one exception 32 or 48 h after rats had received a single dose of daidzein (i.v. or per os). As expected, the daidzein concentrations depended upon dose and administration route. Despite notable differences in the absolute amounts of total daidzein (free plus hydrolyzed conjugates), the levels were usually three- to fivefold higher in liver and kidney than in plasma; in most samples of uteri, the concentrations were similar, or up to twofold higher, than the respective plasma levels. These data point to an uptake and storage of isoflavones and metabolites in tissues. Experimental toxicokinetics appear to be a relevant subject that should be integrated into assessments of toxicological data for endocrine modulators.

Animals↗

PubMed2000

Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotropic activity.

The function of the uterus is regulated by female sex steroids and it is, therefore, used as the classical target organ to detect estrogenic action. Uterine response to estrogens involves the activation of a large pattern of estrogen-sensitive genes. This fact offers the opportunity to analyze the estrogenic activity of xeno- and phytoestrogens, and the mechanisms of their molecular action by a correlation of the uterotropic activity and their ability to modulate the expression of estrogen-sensitive genes. We have analyzed the expression of androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER), clusterin (CLU), complement C3 (C3), and GAPDH mRNA in the rat uterus following oral administration of ethinylestradiol (EE), bisphenol A (BPA), o,p'-DDT (DDT), p-tert-octylphenol (OCT) and daidzein (DAI). A significant stimulation of the uterine wet weight could be observed after administration of all the substances. The activity of all analyzed compounds to stimulate uterine weight was low in comparison to EE. DDT has the highest activity to stimulate uterine weight whereas BPA and DAI turned out to be less potent. The analysis of gene expression revealed a very specific profile of molecular action in response to the different compounds which cannot be detected by judging the uterotropic response alone. A dose dependent analysis revealed that C3 mRNA is already modulated at doses where no uterotropic response was detectable. Although DAI and BPA were very weak stimulators of uterine growth, these substances were able to alter the expression of AR, ER and C3 very strongly. Based on these investigations the analyzed compounds can be subdivided into distinct classes: First, compounds which exhibit a similar gene expression fingerprint as EE (e.g. OCT); second, compounds exhibiting a significant uterotropic activity, but inducing a pattern of gene expression different from EE (e.g. DDT); and third, compounds like BPA and especially DAI which exhibit a very low uterotropic activity, but nevertheless modulate the expression of estrogen-sensitive genes. These findings strongly suggest that the fingerprint of uterine gene expression is a very sensitive tool to investigate estrogenicity of natural and synthetic compounds and offers the possibility to get information in regard to the molecular mechanisms involved in the action of the respective compounds.

Animals↗

PubMed2000

Treatment with the pure antiestrogen faslodex (ICI 182780) induces tumor necrosis factor receptor 1 (TNFR1) expression in MCF-7 breast cancer cells.

Apoptosis induction by the pure antiestrogen faslodex, also known as ICI 182780 (ICI), is associated with an effective down-regulation of Bcl-2 expression in the human breast cancer cell line MCF-7. Recent observations point out that beside members of the Bcl-2 family also the TNFR1 signaling pathway may be involved in apoptosis induction by antiestrogens. In this report we have analyzed the expression of members of the TNFR1 signaling pathway during the apoptotic process induced by the pure antiestrogen faslodex and by tamoxifen (Tam) in MCF-7 breast cancer cells. Treatment with 10(-7) M ICI or 10(-7) M Tam leads to a time dependent increase of TNFR1 and TRADD steady-state mRNA levels in MCF-7 cells. In contrast, Bcl-2 expression was strongly decreased following administration of ICI but only weakly after administration of Tam. Western blot analysis and studies by the use of fluorescence microscopy and flow cytometry revealed a time dependent induction of TNFR1 protein and cell surface expression in MCF-7 cells in response to treatment with ICI. To investigate if TNFR1 is functionally involved in apoptosis induction by antiestrogens, we tested whether TNFR1 blocking antibodies can counteract the growth inhibitory action of Tam and ICI. Coincubation of MCF-7 cells with antiestrogens (ICI or Tam) and blocking TNFR1 antibodies lead to an increase in cell viability. Our results provide evidence for a cross talk between the TNFR1 signaling pathway and antiestrogens during the process of apoptosis induction in MCF-7 breast cancer cells. The superiority of the pure antiestrogen ICI to induce apoptosis in MCF-7 cells may result from its capability to modulate the induction of apoptosis via Bcl-2 as well as TNF-associated signal transduction pathways.

Antigens, CD↗

PubMed1999

Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.

Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression.

Animals↗

PubMed1999

The pure antiestrogen ICI 182780 is more effective in the induction of apoptosis and down regulation of BCL-2 than tamoxifen in MCF-7 cells.

There is increasing evidence that induction of apoptosis by antihormones is an important mechanism in regard to their growth inhibitory action on hormone dependent tumors. In this report we have compared the efficiency of tamoxifen (Tam) and the pure antiestrogen ICI 182780 (ZM) to induce apoptosis in the estrogen dependent breast cancer cell line MCF-7. Clear evidence for induction of apoptosis could be demonstrated after treatment with both antiestrogens. Application of the pure antiestrogen ZM led to a significantly higher induction of apoptosis compared to the partial agonistic compound Tam. The ability of the two compounds to induce apoptosis correlated with their growth inhibitory action. On the molecular level administration of ZM led to a time dependent steady decrease of BCL-2 mRNA and protein. Administration of Tam also initially decreased the expression of BCL-2. In contrast to ZM treatment, BCL-2 expression increased again after 8 h of incubation with Tam. After 96 h Tam treated cells expressed BCL-2 levels nearly as high as untreated cells. In general, ZM decreased BCL-2 levels more effectively than Tam. Our results demonstrate that ZM and Tam possess quantitative and qualitative differences in their ability to down regulate BCL-2 expression. The higher ability of the pure antiestrogen to down regulate BCL-2 expression may explain the superiority of the pure antiestrogen to induce apoptosis and to inhibit the growth of MCF-7 cells.

Apoptosis↗