Alteration of gene expression in rat colon mucosa after exercise.

The development of colon cancer is highly influenced by lifestyle factors such as nutrition and physical inactivity. Detailed biological mechanisms are thus far unclear. The purpose of this study was to investigate the effects of regular treadmill exercise on gene expression in rat colon mucosa. For this purpose, 6-week-old male Wistar rats completed a stress-free voluntary treadmill exercise period of 12 weeks. Sedentary rats served as a control group. In the colon mucosa, steady-state mRNA expression levels of approximately 10,000 genes were compared between both groups by micro-array analysis (MWG rat 10K array). A total of 8846 mRNAs were detected above background level. Regular exercise led to a decreased expression of 47 genes at a threshold-factor of 2.0. Three genes were found to be up-regulated in the exercise group. The identified genes encode proteins involved in signal transduction (n=11), transport (n=8), immune system (n=7), cytoskeleton (n=6), protein targeting (n=6), metabolism (n=5), transcription (n=3) and vascularization (n=2). Among the genes regulated by regular exercise, the betaine-homocysteine methyltransferase 2 (BHMT2) seems to be of particular interest. Physical activity may protect against aberrant methylation by repressing the BHMT2 gene and thus contribute to a decreased risk of developing colon cancer. We have also identified vascular endothelial growth factor (VEGF), angiopoietin-2 (ANG-2) and calcium-independent phospholipase a2 (iPL-A2), all of them with markedly reduced transcript levels in the mucosa of active rats. In summary, our experiment presents the first gene expression pattern in rat colon mucosa following regular treadmill activity and represents an important step in understanding the molecular mechanisms responsible for the preventive effect of physical activity on the development of colon cancer.

Exercise associated genes in rat colon mucosa: upregulation of ornithin decarboxylase-1.

Epidemiology has revealed that physical activity is an important lifestyle factor that reduces the risk of colon cancer. However, the underlying mechanisms of this protective effect have so far not been defined. The aim of this study was to identify molecular targets of physical activity in rat colon mucosa by employing our voluntary exercise model. Twenty male rats underwent a 12-week exercise program, with 9 additional rats serving as a control group. Running distances, body weights and heart weights as measures of physical adaptations were recorded, and changes in mRNA steady-state levels of marker genes involved in vascularization (VEGF, HIF-1 alpha, ODC-1), apoptosis (Bcl-2, PPAR gamma) and prostaglandin synthesis (COX-2) were determined by qRT-PCR. The four housekeeping genes GAPDH, beta-actin, 18S and ALDA served as reference genes. Recorded running distances showed great inter-individual differences resulting in three different groups of low (L-EX, < 2629 m/night; n=5), moderate (M-EX, 3003 - 7458 m/night; n=10) and high (H-EX, > 8314 m/night; n=5) physical activity. The M-EX and H-EX group revealed significant (p<0.05) adaptive changes with an increase in heart mass per kg body weight and a decrease in mean body weight. Amongst the marker genes studied by mRNA expression analysis only ODC-1 appears to be differentially expressed. Its 1.8-fold increased steady-state mRNA level in the H-EX group suggests that synthesis of polyamines may be increased by physical activity. This new finding could provide a link between extensive physical activity and its protective effects on colon cancer development.

Effect of chronic wheel running on the fatty acid composition of phospholipids and triacylglycerols in rat serum, skeletal muscle and heart.

AIM: The purpose of this study was to examine the effects of long-term wheel running on the fatty acid composition of phospholipids (PL) and triacylglycerols (TG) in rat serum, skeletal muscle (soleus and extensor digitorum longus) and heart. METHODS: To this end, the relevant tissues of 11 trained male Wistar rats were compared with those of 14 untrained ones. RESULTS: There were several significant differences between the two groups regarding the concentrations and percentages of individual fatty acids in serum PL and TG, with most differences appearing in the fatty acid distribution of PL. Monounsaturated fatty acids of muscle PL were significantly lower in the trained rats. Estimated elongase activity was significantly higher, whereas Delta(9)-desaturase activity was significantly lower in the trained muscles. Monounsaturated fatty acids of PL were also significantly lower in the trained hearts. The fatty acid composition of PL in the skeletal muscles and the heart adapted to training in a comparable manner, whereas most of the changes in the fatty acid profile of TG were tissue-dependent. Judging from the magnitude of the effect sizes and the percentage differences between trained and untrained animals, there were many large effects of chronic exercise on the fatty acid composition of the tissues examined. CONCLUSION: Long-term wheel running modified the fatty acid profile of PL and TG in rat serum, skeletal muscle and heart, and could thus be considered as a modulator of tissue fatty acid composition.

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.

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.

Phagocytic function in cyclists: correlation with catecholamines and cortisol.

Flow cytometer measurements were made of the basal variations in peripheral blood functional monocytes and granulocytes over the course of a training season (January to November) of a cycling team. Parallel determinations were made of plasma concentration of catecholamines (chromatography) and cortisol (RIA) in a search for neuroendocrine markers. The results showed the greatest phagocytic capacity to occur in the central months (March, May, and July), coinciding with the greatest number and highest level of competitive events with good correlation with a peak in epinephrine during these months (r(2) = 0.998 for monocytes and r(2) = 0.674 for granulocytes). No good correlations were found between phagocytosis and norepinephrine or cortisol. The highest values for phagocytosis and epinephrine concentration were found in May. These results suggest that blood epinephrine concentration could be a good neuroendocrine marker of sportspeople's phagocytic response.

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.

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.

Comparative assessment of endocrine modulators with oestrogenic activity: I. Definition of a hygiene-based margin of safety (HBMOS) for xeno-oestrogens against the background of European developments.

A novel concept - the hygiene-based margin of safety (HBMOS) - is suggested for the assessment of the impact of potential endocrine modulators. It integrates exposure scenarios and potency data for industrial chemicals and naturally occurring dietary compounds with oestrogenic activity. An HBMOS is defined as a quotient of estimated daily intakes weighted by the relative in vivo potencies of these compounds. The Existing Chemicals Programme of the European Union provides Human and Environmental Risk Assessments of Existing Chemicals which include human exposure scenarios. Such exposure scenarios, along with potency estimates for endocrine activities, may provide a basis for a quantitative comparison of the potential endocrine-modulating effects of industrial chemicals with endocrine modulators as natural constituents of human diet. Natural phyto-oestrogens exhibit oestrogenic activity in vitro and in vivo. Important phyto-oestrogens for humans are isoflavones (daidzein, genistein) and lignans, with the highest quantities found in soybeans and flaxseed, respectively. Daily isoflavone exposures calculated for infants on soy-based formulae were in the ranges of 4.5-8 mg/kg body wt.; estimates for adults range up to 1 mg/kg body wt. The Senate Commission on the Evaluation of Food Safety (SKLM) of the Deutsche Forschungsgemeinschaft has also indicated a wide range of dietary exposures. For matters of risk assessment, the SKLM has based recommendations on dietary exposure scenarios, implying a daily intake of phyto-oestrogens in the order of 1 mg/kg body wt. On the basis of information compiled within the Existing Chemicals Programme of the EU, it appears that a daily human exposure to nonylphenol of 2 microg/kg body wt. may be a worst-case assumption, but which is based on valid scenarios. The intake of octylphenol is much lower, due to a different use pattern and applications, and may be neglected. Data from migration studies led to estimations of the daily human uptake of bisphenol A of maximally 1 microg/kg body wt. On the basis of comparative data from uterotrophic assays in rats, with three consecutive days of oral applications involved, and taking the natural phyto-oestrogen daidzein as reference (= 1), relative uterotrophic activities in DA/Han rats follow the sequence: daidzein = 1; bisphenol A = 1; p-tertoctylphenol = 2; o, p'-DDT = 4; ethinyl oestradiol = 40,000. The derived values from exposure scenarios, as well as these relative potency values and bridging assumptions, led to calculations of HBMOS as a quantitative comparison of potential endocrine-modulating effects of industrial chemicals with those of natural constituents of human diet. HBMOS estimates for nonylphenol ranged between 250 and 500, dependent on bridging assumptions, and around 1000 for bisphenol A. The derivations of HBMOS were in full support of the conclusions reached by the SKLM of the Deutsche Forschungsgemeinschaft. The estimated HBMOS values for the industrial chemicals (nonylphenol, bisphenol A) appear sufficiently high to ensure the absence of a practical risk to human health under the present exposure conditions.

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.

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.

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.

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.

The antiovulatory potential of progesterone antagonists correlates with a down-regulation of progesterone receptors in the hypothalamus, pituitary and ovaries.

These studies analyze the regulation of progesterone receptors (PRs) in central and peripheral tissues with the aim of further understanding mechanistically the inhibition of ovulation by progesterone antagonists (PA). Therefore, it was of interest to investigate the influence of the progesterone receptor antagonist, Onapristone (ON), on PRs in the ovary, pituitary (PT), and hypothalamus (HYP), since ON effectively inhibits ovulation in rats. For this study PMSG/hCG-primed immature and adult female rats were treated with ON. Immunohistochemistry was used for the detection of PRs. Progesterone (P4) and estradiol (E2) levels were determined by RIA. PR expression in the ovaries of immature rats was not detectable until after hCG administration. In these animals, ON caused a reduction in the staining intensity of PR in the tertiary follicles at the time when the preovulatory P4-surge was inhibited (6 h post hCG). Adult rats treated for 15 days with ON showed a decreased PR expression in PT and HYP. At this time (proestrus, 7 p.m.) the P4 and E2 levels are significantly lowered. These results suggest that after treatment with ON the expression of PR is reduced in the ovary, PT and HYP. The regulation of PR in the ovary seems to be less dependent on estrogens than on LH. Thus, it is conceivable that the reduced PR expression after ON treatment may be a result of decreased LH sensitivity in the ovary. In the pituitary and hypothalamus, PR expression is stimulated by estrogens and progesterone, and therefore the fall in the P4 and E2 levels in ON-treated animals may be responsible for the reduced PR expression in PT and HYP, and may contribute to the antiovulatory effect of ON. We therefore conclude that the mechanism of the antiovulatory potency of progesterone antagonists is based on a reduced preovulatory P4-production and PR expression in the ovary and also on the down-regulation of PR in the anterior pituitary and hypothalamus.

In vitro test systems for the evaluation of the estrogenic activity of natural products.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. There is evidence that the consumption of some of these phytoestrogens may have beneficial effects but it also seems possible that others may act as endocrine disrupters. For this reason there is a need to characterise the estrogenic potency of these substances. In vitro test systems offer the possibility to screen compounds very efficiently. Routinely in use and widespread for the determination of estrogenicity are: (I) receptor binding assays, (II) cell-proliferation assays (E-screens), (III) reporter gene assays, and (IV) the analysis of the regulation of endogenous estrogen sensitive genes in cell lines. The basis of all these test systems are molecular mechanisms which are involved in the classical estrogen action. In addition, in the last years several test systems for the investigation of non-classical estrogenic effects have been established. An example for such an effect is the modulation of the expression of interleukin-6, a cytokine that appears to be a key molecule in the osteoporotic process, by estrogens. Summarising the advantages and the issues of all presented in vitro test systems, it seems to be evident that only the analysis of results obtained in a combination of several in vitro test systems may validly predict effects in vivo.

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.

Functional activity of immune cells in female MS-patients.

Since the well documented immunological disturbances in patients suffering from multiple sclerosis are especially attributed to T-cells we felt it expedient to look also for the functional activity of lymphocytes, monocytes, granulocytes and natural killer cells in the peripheral blood of female MS-patients (n = 17) and healthy controls (n = 17). While MS-patients showed decreased levels of total lymphocytes, CD2(+)- and CD8(+)-cells the percentage of HLA-DR(+)-monocytes was increased, indicating a high activity level of these immune cells, whereas we could find no differences in the functional tests of monocytes, granulocytes and natural killer cells between MS-patients and healthy controls. The percentage of HLA-DR(+)-T-lymphocytes increased with the duration of the illness and support the stronger consideration of clinical staging.

The antiovulatory effect of the antiprogestin onapristone could be related to down-regulation of intraovarian progesterone (receptors).

The present study was undertaken to investigate intraovarian mechanism(s) for the antiovulatory effect of Onapristone (ON), because antiprogestins possessing the same antiprogestational activity and inhibiting the preovulatory LH surge to the same extent differ in their antiovulatory potency. Ovulation was induced by treating immature female rats with pregnant mare serum gonadotropin (PMSG) for folliculogenesis and hCG for the induction of ovulation. The animals were treated twice with ON (200 mg/kg 42 h and 48 h after PMSG) and killed at different times. The ovulation rate was assessed by counting the number of ova in the fallopian tubes and uteri. Blood and ovaries were collected for radioimmunoassay (RIA) of steroid hormones and histological analysis for 3beta-hydroxysteroid dehydrogenase (3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), progesterone (PR), estrogen (ER) and androgen (AR) receptors. Treatment with ON totally blocked ovulation and the progesterone (P4) surge was significantly diminished in comparison to the control (6-8 h post-hCG), whereas androgen levels remained unaffected. The decreased P4 concentrations correlated well with a reduced staining intensity of 3beta-HSDH in granulosa cells of tertiary follicles. Moreover, we observed a down-regulation of PR in granulosa cells of tertiary follicles. Additionally, in secondary and tertiary follicles the expression of AR between 0 and 6 h after hCG was reduced. These results suggest that the antiovulatory effect of the antiprogestin ON is related to down-regulation of intraovarian progesterone, caused by attenuated 3beta-HSDH activity and PR expression. One can thus assume that intraovarian P4 is an important factor for the induction of ovulation. An effect of ON on the staining intensity of 17beta-HSDH in theca and granulosa cells could not be observed at any time. In conclusion, the inhibition of ovulation induced by the antiprogestin, ON, could be related to decreased intraovarian progesterone production through reduced 3beta-HSDH activity and the down-regulation of PR.

Progesterone receptor repression by estrogens in rat uterine epithelial cells.

Measurements performed using cell lines or animal tissues have shown that the progesterone receptor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of estrogens on the PR levels in the individual cell types of the target organs uterus and breast. In the uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and surrounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture emerged when intact rats were treated with the pure estrogen receptor antagonist, ZM 182780 (10 mg/kg/d). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myometrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we found that in the epithelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced mammary carcinomas the PR expression was induced by estrogens and was blocked by the pure antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diverging effects on PR expression in different cell types even within the same organ.

The antiovulatory activity of progesterone antagonists is not correlated to their antiprogestational potency in the rat.

Progesterone antagonists often differ in regard to their potency to block ovulation in rats although they may possess similar 'antiprogestational' (abortive) activity. Therefore, the questions arose as to: (a) whether antiovulatory and antiprogestational effects (on endometrial and mammary gland parameters) of antiprogestins correlate at all; and (b) which mechanism(s) may be responsible for their ability to abolish ovulation. To answer these questions we set out to compare the influences of two progesterone antagonists, Onapristone (ON) a very potent and ZK 136798 only a weak inhibitor of ovulation, to assess changes on the one hand on typical progestational actions and on the other hand on factors known to regulate ovulation. For this purpose immature PMSG/hCG primed and adult female rats and infantile female rabbits were treated either with ON, ZK 136798 or vehicle in different treatment schedules. In these investigations ON and ZK 136798 showed similar antiprogestational activities on the progesterone-induced development of mammary glands (rats) and the secretory transformation of endometrium (rabbits). ON blocked an induced or a spontaneous ovulation, whereas ZK 136798 only revealed a very weak antiovulatory effect. Both ON and ZK 136798 stimulated basal levels of LH, estradiol, and testosterone, whereas the preovulatory LH surge was decreased to the same extent. Interestingly, in contrast to ZK 136798, ON reduced the preovulatory increase in progesterone secretion. These results clearly indicate: (a) that antiovulatory potency and antiprogestational activity may not be correlated in the rat; and (b) that decreased preovulatory levels of progesterone following treatment with ON may play an important role in intraovarian mechanism(s) contributing to a blocking of ovulation.