Circadian modulation of GABA-mediated cortical inhibition.

Circadian rhythms exert powerful influence on various aspects of human physiology and behavior. Here, we tested changes of human cerebral cortex excitability over the course of the day with transcranial magnetic stimulation (TMS). At different times of the day, intracortical and corticospinal excitability of the primary motor cortex (M1) was evaluated in 15 healthy subjects by TMS of left M1. While motor thresholds, short-interval intracortical inhibition and facilitation and input/output curves remained unchanged, we found that a specific form of γ-aminobutyric acid (GABA)-mediated intracortical inhibition, revealed by long-interval intracortical inhibition and cortical silent periods, progressively decreased during the course of the day. Additional experiments demonstrated that morning inhibition persisted irrespective of previous sleep or sleep deprivation. Corticotropin-releasing hormone (CRH) infusions in the evening lead to morning cortisol levels but did not restore levels of morning inhibition, whereas suppression of endogenous CRH release by repeated oral dexamethasone intake over 24 h prevented morning inhibition. The findings suggest a specific modulation of GABAergic motor cortex inhibition within the circadian cycle, possibly linked to the CRH system, and may indicate a neurobiological basis for variable neuroplasticity over the course of the day.

20-OH-ecdysone prevents hot flushes in ovariectomized rats.

Hot flushes are due to the lack of estrogens and are the most characteristic climacteric complaints. Hormone replacement therapy was the standard treatment but now its use is limited because of side effects. Need therefore arises to search for non-estrogenic alternatives. The molting hormone 20-beta-hydroxyecdysone (Ecd) is produced by several plants including spinach and has no estrogenic or androgenic properties but enhances GABAergic effects in neurons. Since GABAergic compounds can ameliorate hot flushes, we investigated the effects of Ecd on subcutaneous body temperature of intact and ovariectomized (ovx) rats. The subcutaneous body temperature was recorded at 5-min intervals over a period of 3 hours. Rats were then ovx, and skin temperatures were recorded after an acute intravenous (5 mg) and during subchronic and chronic oral application of Ecd (73 mg/animal/day). For additional control purposes, a group of ovx rats received food containing estradiol-17 ß (E2). Skin temperature in individual ovx animals fluctuated largely with peaks (hot flushes) occurring every 20-40 minutes. Following the i.v. treatment with Ecd, skin temperature dropped by more than 1 °C, an effect much larger than in the controls. One and two weeks later, hot flushes were only seen in ovx controls but not in intact, E2-, or Ecd-treated animals. As a consequence, E2 and Ecd intake significantly (p < 0.05) reduced the mean temperature in ovx rats during the various time points of the study. These results suggest that Ecd is efficient to prevent hot flushes in ovx rats.

Long-term treatment of ovariectomized mice with estradiol or phytoestrogens as a new model to study the role of estrogenic substances in the heart.

Epidemiological data reveal that the overall risk for heart disease is lower for premenopausal women compared to age-matched men. However, the beneficial effect for the female sex is lost upon menopause. Thus, it has been suggested that estrogens convey the protective effect for the female sex against heart disease. Numerous natural plant products, i.e., phytoestrogens (PE), interfere with or alter the development or function of the endocrine system. Although PEs have been studied intensively with regard to the effects on the reproductive organs, such as the uterus or mammary gland, surprisingly little data are available about the effects of PEs on the heart. Here, we conducted a long-term study with ovariectomized mice to examine putative estrogenic effects of the PEs genistein (GEN), resveratrol (RES), and equol (EQ), using estradiol (E2) as a reference compound on heart size, morphology, and cardiac gene expression. We report for the first time significant changes in these parameters by GEN and E2. Changes in the size of cardiomyocytes were observed by GEN and E2. In line with these observations, cardiac expression of insulin-like growth factor 1 ( IGF1) was significantly induced by both GEN and E2. Thus, we speculate that endocrine active compounds, like the isoflavone GEN, which is used as a food additive or as a drug for the treatment of menopausal symptoms, may directly affect heart function.

Do estrogen and alendronate improve metaphyseal fracture healing when applied as osteoporosis prophylaxis?

Osteoporosis is accompanied by predominantly metaphyseal fractures with a delayed and qualitatively reduced healing process. This study addressed the question of whether fracture healing in the context of osteoporosis prophylaxis is improved with estrogen (E) or alendronate (ALN). Thirty-six ovariectomized and 12 sham-operated 12-week-old rats received soy-free (osteoporotic C, sham), E-, or ALN- supplemented diets. After 10 weeks, a metaphyseal tibia osteotomy and standardized T-plate fixation were performed. After a 5-week healing process, the fracture callus was evaluated qualitatively by biomechanical bending test and quantitatively in microradiographic sections. The time course of callus formation was examined using fluorochrome-labeled histological sections. Administration of E improved the biomechanical properties of callus (stiffness [N/mm]: sham: 110.2 + or - 76.07, C: 41.28 + or - 33.70, E: 85.72 + or - 47.24, ALN: 72.07 + or - 34.68). The resistance to microfracturing seen in E-treated animals was significantly enhanced and even superior to sham (yield load [N] sham: 27.44 + or - 9.72, C: 21.04 + or - 12.47, E: 42.85 + or - 13.74(Delta), ALN: 25.28 + or - 6.4(.)) (* P < 0.05 vs. sham group, (Delta) P < 0.05 vs. C group, (*) P < 0.05 vs. E group). Trabecular bone in particular was improved, indicating the presence of physiological endosteal bridging (Tr.Dn [%] sham: 10.53 + or - 18.9, C: 1.01 + or - 0.14, E: 24.13 + or - 34.09(Delta), ALN: 3.99 + or - 8.3(.)). ALN did not help bone healing, as shown by mechanical tests. Compared to the C group, statistically, ALN did not show worse properties. The induction of callus formation under ALN treatment was slightly delayed (Tt.Cl [mm(2)] sham: 3.68 + or - 0.66, C: 3.44 + or - 0.42, E: 3.69 + or - 0.58, ALN: 3.06 + or - 0.56). Osteoporotic metaphyseal fracture healing was qualitatively and quantitatively improved by E prophylaxis. The process of fracture healing occurred nearly physiologically (shamlike). Notably, ALN hardly improved metaphyseal callus properties when assessed as osteoporosis prophylaxis, but to a lesser extent than E.

Plant-derived alternative treatments for the aging male: facts and myths.

Soy- or red clover- derived products containing isoflavones have been amply studied in climacteric and postmenopausal women, and confusing contradicting results have been published. The beneficial effects on climacteric complaints, cholesterol and the development of osteoporosis are marginally at best and there are no uterine and mammary safety studies. In males, however, isoflavones may protect the prostate to make them less prone to develop cancer. Cell biological and animal experimental data support this notion. Clinical data about possible beneficial effects on cholesterol or in the bone are largely missing. Hence, soy or red clover products containing the mild estrogenic isoflavones with a slightly higher affinity to the estrogen receptor of the beta in comparison to the alpha subtype may prove to have some beneficial effects in males.

Changes in the histomorphometric and biomechanical properties of the proximal femur of ovariectomized rat after treatment with the phytoestrogens genistein and equol.

The isoflavonoids found in soy have attracted great interest as dietary phytoestrogens that might be effective for postmenopausal hormone replacement therapy. Special attention has been devoted to the hormonal effects of various isoflavonoids, like genistein (GEN) and daidzein's (DAID) potent metabolite, equol (EQ). Here we aimed to investigate the short-term effects of genistein and equol on the proximal femur of ovariectomized (OVX) rats. Forty-eight, 3-month-old female Sprague-Dawley rats were ovarectomized; after eight weeks the bilateral osteotomy and osteosynthesis (OS) of their tibiae was performed and the rats were randomly divided into the following four groups: OVX control group (C), treated with estradiol-17beta (E2) -benzoate (E; daily intake 0.086 mg/d per animal), genistein (GEN; daily intake 12.7 mg/d per animal) and equol (EQ; daily intake 4.65 mg/d per animal). At 5 weeks postoperatively (OS), the breaking test was performed on the trochanteric region of femur. Additionally, histomorphometric assessment, and trabecular and cortical bone microstructure analyses were performed. The relative gain of body weight (BW) in the EQ (24 %) group was significantly (p < 0.05) lower than in the C (33 %) and GEN (30 %) groups. After treatment for 5 weeks, the maximal load (F(max)) and yield load (yL) were higher (p < 0.05 for the weight-adapted results) in the E (188.4 N resp. 113.1 N) and EQ (177.3 N resp. 112 N) groups as compared to C (162.8 N resp. 109.1 N) and GEN (165.7 N resp. 108.8 N). In the histomorphometric tests the E- (trabecular area (Tb.Ar) = 74.93 %, trabecular nodes/mm(2) (N.Nd/mm(2)) = 48.65) and EQ-treated (Tb.Ar = 63.13 %, N.Nd/mm(2) = 43.72) animals showed significant improvement with regard to Tb.Ar and trabecular connectivity (N.Nd./mm(2)) in comparison to C (Tb.Ar = 46.84, N.Nd/mm(2) = 31.86) and GEN (Tb.Ar = 48.22 %, N.Nd/mm(2) = 34.15). There were no differences in relative cortical width (Ct.Wi) among the four groups. The treatment with EQ resulted in improved biomechanical and histomorphometric properties as compared to the treatment with GEN. Thus, of the studied substances, EQ seems to be a possible alternative to hormone replacement therapy, but further studies are needed.

Absence of positive effect of black cohosh (Cimicifuga racemosa) on fracture healing in osteopenic rodent model.

The healing of predominantly metaphyseal fractures in postmenopausal osteoporosis is delayed and comparatively poor. Due to the potential side effects of HRT, natural alternatives are appealing. The aim of this study was to determine whether Cimicifuga racemosa extract BNO 1055 improves metaphyseal fracture healing in severe osteopenic bone in rats. Thirty-three 12-week-old female rats developed severe osteopenia during 10 weeks after ovariectomy. After metaphyseal tibial-osteotomy and standardized T-plate-osteosynthesis, the healing periods in ovariectomized rats (C), 17-α-estradiol (E) and Cimicifuga racemosa (CR) supplemented diets were assessed for 35 days. Changes in callus morphology were evaluated qualitatively by biomechanical testing and quantitatively in microradiographies and fluorochrome-labeled histological sections. The CR-supplementation slightly improved callus quality and trabecular bone formation. It significantly enhanced the endosteal callus density compared to C group (Cl.Dn.e C: 59.08 ± 21.89, E: 45.95 ± 18.39, CR: 60.85 ± 18.66*), though most of the other morphological parameters examined showed no improvement. The time course of fracture healing did not change due to CR. Estrogen-supplementation enhanced the biomechanical properties of the fracture site. Trabecular bone was improved indicating the physiological endosteal healing process. The CR-supplementation did not exhibit positive effects in severe (senile) osteopenic fracture healing as seen in early (postmenopausal) osteoporosis in rats. Callus formation was slightly improved under CR. Estrogen improved fracture healing in severe osteopenic bone, while the extent of callus formation played a minor role.

Effects of estradiol benzoate, raloxifen and an ethanolic extract of Cimicifuga racemosa in nonclassical estrogen regulated organs of ovariectomized rats.

The special extract of Cimicifuga racemosa (CR) BNO 1055 was shown to have bone protective effects without exerting estrogenic effects in the uterus or mammary gland. Whether the effects of CR BNO 1055 would be exerted in other organs that also express estrogen receptors (ERs) but in which the effects of estrogens and of the selective estrogen receptor modulator raloxifen (Ral) were not thoroughly studied was therefore investigated in the present contribution. Rats were ovariectomized (ovx) and their food immediately substituted with estradiol benzoate (EB), Ral or 2 doses of CR BNO 1055 for 3 months. Expressions of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and of insulin-like growth factor-1 (IGF-1) genes were determined in the vagina, liver, thyroid gland, lung, spleen, colon and kidney by means of quantitative RT-PCRs. Body weights in all treatment groups were significantly reduced and uterine weights in the EB treated animals were largely and in the Ral treated animals slightly but significantly increased. CR BNO 1055 was without effects in the uterus. We tested 3 genes: ERalpha gene expression was significantly reduced in the vagina, liver and kidney and remained unaffected in all other organs with the exception of the thyroid gland where ERalpha gene expression was stimulated by EB, Ral had--if any--similar effects in these organs. The CR extract BNO 1055 was devoid of any effect on ERalpha gene expression. ERbeta gene expression was suppressed in the vagina and colon by EB and this effect was shared by Ral in the colon. In the thyroid, EB and Ral stimulated ERbeta gene expression. Expression of IGF-1 gene was stimulated by EB and CR BNO 1055 in the vagina and kidney and inhibited by EB and Ral in the liver. No effects were observed by CR BNO 1055 in these organs. The effects of Ral, if occurring, were similar to those of EB while CR BNO 1055 was ineffective in all organs but the vagina. In the colon, reduced ERbeta gene activity may augment ERalpha mediated effects. In all other organs the effects of ER await further investigation. The CR BNO 1055 did not show any activity pattern which would be similar to the pattern observed under EB or Ral. Therefore the observed effects of CR BNO 1055 in these organs are most likely not estrogenic in nature.

Effects of estrogen receptor alpha- and beta-selective substances in the metaphysis of the tibia and on serum parameters of bone and fat tissue metabolism of ovariectomized rats.

The functions of estrogen receptors (ER) alpha and beta (ER-alpha and beta) in bone and fat tissue are not precisely described. Therefore we studied the effects of a specific ERalpha and ERbeta agonist in bone and fat of ovariectomized (ovx) rats and compared them with the effects of estradiol (E2). Animals were s.c. injected for 4-weeks with 3 doses of the ERalpha agonist 16alpha-LE2 or the ERbeta agonist 8beta-VE2 or with E2. The intermediate doses were antagonized by an additional daily treatment with ICI (1.53mg). Bone and fat parameters were evaluated by quantitative computer tomography (qCT). Estrogen regulated hormones were also measured. Uterine weights were stimulated; serum LH and leptin levels suppressed E2 and the ERalpha agonist. Density of the cancellous metaphyseal structures of the tibia was reduced in the controls which was prevented by E2 and the ERalpha agonist. Endosteal surface, endosteal, periosteal circumferences and fat depots were largest in the controls and the ERbeta treated animals and lowest in the E2 and the 16alpha-LE2 injected ovx rats. Osteocalcin and the CrossLaps were highest in the ovx controls and reduced by E2 and the ERalpha agonist. Serum osteocalcin was stimulated by the ERbeta agonist. The strain strength index (SSI) in relation to the bodyweight - an indicator of bone elasticity - was lowest in controls and increased dose dependently in the E2 and in the ERalpha treated animals. Most effects in the uterus, serum and bone were antagonized by ICI. Most effects in the bone and fat were exerted by mechanisms involving the ERalpha but the ERbeta agonist appears to stimulate osteoblasts.

Effects of dietary equol administration on the mammary gland in ovariectomized Sprague-Dawley rats.

OBJECTIVE: To evaluate the effects of dietary equol, a metabolite of soy-derived daidzein or formononetin present in red clover, on the mammary gland of ovariectomized Sprague-Dawley rats. DESIGN: Sixty ovariectomized rats were divided into five groups (n = 12) and fed soy-free chow with the addition of equol (50 mg/kg chow and 400 mg/kg chow) or estradiol-3 benzoate (E2B) (4.3 mg/kg chow and 17.3 mg/kg chow). The control group received soy-free chow only. After 3 months animals were killed, blood was collected, and the mammary glands were removed for histological and immunohistochemical evaluation. RESULTS: Equol and E2B treatment significantly increased serum equol and 17beta-estradiol concentrations, respectively. Serum prolactin in animals treated with high-dose equol was also significantly higher than in the controls. Animals treated with high-dose equol had a significantly higher number of terminal ducts and type II lobules compared with controls. This was also apparent in animals treated with low- and high-dose E2B, but a higher number of type I lobules also was seen. Compared with controls, animals treated with high-dose equol had a significantly higher percentage of proliferating cell nuclear antigen-positive cells in terminal ducts and type II lobules. The percentage of progesterone receptor-positive cells in animals treated with high-dose equol was significantly higher only in type II lobules. In animals treated with low- and high-dose E2B, the percentage of proliferating cell nuclear antigen- and progesterone receptor-positive cells was significantly higher in all the mammary structures. Low-dose equol did not have any effects on the parameters listed above. CONCLUSIONS: High-dose dietary equol administration to ovariectomized rats exerts clear mammotropic effects.

Effects of a 5-day treatment with vinclozolin on the hypothalamo-pituitary-gonadal axis in male rats.

Vinclozolin (VZ), a potent antiandrogenic fungicide, is known to interfere with male reproductive function. Little data are currently available regarding possible impacts of VZ on brain function, particularly neuroendocrine activity and regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Therefore, we examined the effects of VZ on gene expression in the brain (MBH/ME, MPOA/AH, striatum, hippocampus), pituitary, prostate, seminal vesicles, and epididymis of 4-month-old male rats treated daily by gavage for 5 days with VZ (150 mg/kg body weight/day). Alterations in levels of serum hormones and gene expression were determined by RIA and qRT-PCR, respectively. Our results revealed that (i) VZ decreases epididymis weights, increases serum levels of LH and T, and decreases serum TSH and total T(4) levels; (ii) VZ affects the hypothalamic expression of both estrogen receptor (ERs) subtypes, ERalpha and ERbeta; (iii) in the extrahypothalamic brain areas, VZ alters expression of ERs and androgen receptor (AR); (iv) in the pituitary, VZ up-regulates expression of the GnRH receptor, LHbeta, alpha-subunit, and TERP-1/-2; (v) in the ventral prostate, VZ increases and decreases levels of AR and ERbeta mRNA, respectively; (vi) in the seminal vesicles, VZ increases levels of AR and ERalpha mRNA expressions; (vii) in the epididymis, VZ up-regulates AR and ERbeta mRNA expression. These results indicate that in vivo VZ is not a 'pure' antiandrogen, since it exerts mixed AR antagonistic/ERs agonistic actions observed at the levels of mRNA expression of selected androgen- and estrogen-regulated genes in the CNS, pituitary, and male accessory sex organs.

Pharmacokinetics and metabolism of benzophenone 2 in the rat.

Twelve derivatives of benzophenone (BP1-BP12) are widely used as UV-screens to protect industrial products from light induced damage. There is growing public concern about industrially produced chemicals that might interfere with hormonal signalling pathways, thus having potential adverse effects on human health. The derivative 2,2',4,4'-tetrahydroxybenzophenone (BP2) which is used in cosmetic products and in packaging materials, was previously shown to be an estrogenic endocrine active chemical (EAC). While the metabolisation of BP3 has been analyzed in vivo, according to our knowledge little is known about the pharmacokinetics of BP2. Therefore we performed a dose-response experiment with 5 dosages of BP2 which was applied per gavage to adult ovariectomised (ovx) rats for 5 days. Serum samples were analyzed via HPLC. Metabolites were further identified by Helix pomatia glucuronidase treatment and subsequent ion-trap-mass spectrometry. Additionally we analyzed the time dependent metabolisation and excretion of BP2 in a kinetic study. The parent compound BP2 is metabolised to glucuronide - and sulfate-conjugates. In the serum maximum levels of BP2, BP2-glucuronide and BP2-sulfate were observed already 30 min after BP2 application while highest concentrations of BP2 and its metabolites in urine were measured 120 min after treatment. It is suggested that this biotransformation occurs via a first-pass effect in the gut wall or the liver. Despite this rapid metabolisation and excretion, the amount of unconjugated BP2 was sufficient to induce a dose dependent estrogenic effect in the uterotrophic assay.

Effects of black cohosh extract on body weight gain, intra-abdominal fat accumulation, plasma lipids and glucose tolerance in ovariectomized Sprague-Dawley rats.

UNLABELLED: Extracts of the black cohosh (Actaea/Cimicifuga racemosa (CR)) have long been used to treat estrogen deficiency symptoms in women after menopause. Recent data from randomized controlled studies have shown that CR consumption alleviates "hot flushes" and due to the lack of uterotropic effects can be a safe alternative to estrogen replacement therapy. OBJECTIVE: To evaluate the effects of dietary CR extract consumption on body weight (BW) gain, intra-abdominal fat (IAF) accumulation, plasma leptin, lipids and glucose tolerance in ovariectomized rats and to compare them with the effects of 17beta-estradiol. DESIGN: Twenty-seven female Sprague-Dawley rats were ovariectomized and fed soy-free chow with the addition of estradiol-3 benzoate (E2B) (10mg/kg, n = 10) or CR BNO 1055 extract (6.67 g/kg, n = 9). The control group (n = 8) received soy-free chow only. Weight and food intake were recorded once a week. After 6 weeks, intra-abdominal fat was measured using computer tomography and the intraperitoneal glucose tolerance test was performed. In the seventh week of the experiment animals were sacrificed, blood was collected for plasma and uteri were removed. RESULTS: Dietary CR BNO 1055 extract had no effects on uterine mass but significantly reduced serum lutenizing hormone (LH) levels (P < 0.05). Although, the average weekly food consumption throughout the experiment (calculated in g/kg of BW) did not differ between our studied groups, E2B or CR BNO 1055 treated animals gained less weight and had significantly less IAF accumulation compared to control animals (P < 0.05). E2B treatment also decreased plasma total (T-,) high-density lipoprotein (HDL-) and low-density lipoprotein (LDL)-cholesterol (P < 0.05). Plasma T-Ch levels in CR BNO 1055 treated animals did not differ from the controls whereas LDL-Ch levels were significantly higher and plasma triglycerides (TG) significantly lower (P<0.05). In the glucose tolerance test, the area under the curve (AUC) was significantly smaller in the E2B treated animals compared to controls (P<0.05). AUC in CR BNO 1055 treated animals did not differ significantly from the controls (P>0.05). Nevertheless, fasting plasma insulin (FPI) levels were significantly lower in E2B and CR BNO 1055 treated animals (P<0.05). CONCLUSIONS: In OVX rats, CR BNO 1055 extract consumption decreases enhanced pituitary LH secretion, attenuates body weight gain and IAF accumulation, lowers FPI and has no effects on uterine mass. The effects on plasma lipids seem to be more complex and are characterized by an increase of LDL-Ch and decrease of TG levels which is in contrast to the effects of estrogen.

Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?

OBJECTIVES: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed. METHODS: Review of selected publications since 1980 and summary of unpublished own results of the authors. RESULTS: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect. CONCLUSION: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

Dietary quercetin does not affect pituitary lutenizing hormone (LH) expression and has no uterotropic effects in ovariectomized Sprague-Dawley rats.

INTRODUCTION: The aim of this study was to investigate the potency of LH suppression and the uterotrophic effects of quercetin, a flavonoid widely present in our diet which in vitro has been shown to posses estrogenic properties. METHODS: Fifty-nine female Sprague-Dawley (SD) rats were ovariectomized (ovx) and fed with soy-free rodent chow with the addition of quercetin or estradiol-3 benzoate (E2B). Quercetin was added to the rodent chow at the dose of 200mg/kg (n=12) and 1000 mg/kg (n=11) which on average corresponded to 3.55 mg and 18.42 mg per animal per day, respectively, and E2B at the dose of 4.3mg/kg (n=12) or 17.3mg/kg (n=12) which corresponded to 0.07 mg and 0.20 mg per animal per day, respectively. The control group (n=12) received soy-free chow only. After three months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHbeta and uterine insulin like growth factor (IGF)-1, progesterone receptor (PR) and complement 3 protein (C3) mRNA levels were measured. Additionally, the in vitro binding capacity of quercetin with a porcine cytosolic ER preparation was evaluated. RESULTS: In contrast to E2B, dietary quercetin did not decrease pituitary LH expression, had no effects on uterine weight and uterine expression of estrogen regulated genes. The binding capacity of quercetin with the ERs was also 35000-fold lower compared with 17beta-estradiol (E2). CONCLUSION: Our study shows that quercetin does not show any estrogenic effects in the pituitary and the uterus of the ovx SD rats.

The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase.

Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H(2)O(2) (10 mumol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T(4) was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.

Isoflavones--safe food additives or dangerous drugs?

The sales volume of products containing isoflavone has increased since the publication of the Women's Health Initiative. The many apparently contradictory results published on the effects of isoflavones on a variety of estrogen-regulated organs point to both beneficial as well as adverse effects on human health. It is of particular importance that psychovegetative climacteric complaints such as hot flushes are, if at all, only slightly influenced by isoflavones. The substances appear to have weak anti-osteoporotic effect. Their anti-atherosclerotic action is debatable, as not all authors find any beneficial effect on lipids. Most importantly, there is dispute as to whether isoflavones derived from soy or red clover have negative, positive or any effect at all on the mammary gland or endometrium. It is beyond any doubt that soy products may have cancer preventing properties in a variety of organs including the mammary gland. However, these properties may only be exerted if the developing organ was under the influence of isoflavones during childhood and puberty. This may also explain the often quoted "Japanese Phenomenon", the fact that breast cancer occurs to a lesser extent in Japanese women. When administered to isoflavone "inexperienced" women at the time of menopause, the phytoestrogens appear to share the same effects as estrogen used in classical preparations for hormone replacement therapy, i.e. they may stimulate the proliferation of endometrial and mammary gland tissue with at present unknown and unpredictable risk to these organs. Therefore, the following question arises for the clinician: Why should soy or red clover products containing isoflavone be recommended, if the positive effects are only negligible but the adverse effects serious?

Effects of chronic genistein treatment in mammary gland, uterus, and vagina.

BACKGROUND: The isoflavone genistein (GEN) is found in soy (Glycine max) and red clover (Trifolium pratense). The estrogenic activity of GEN is known, and it is widely advertised as a phytoestrogen useful in alleviating climacteric complaints and other postmenopausal disorders. Knowledge of effects of long-term administration of GEN in laboratory animals is scarce, and effects in the uterus and mammary gland after long-term administration have not been studied. The uterus and mammary gland are known to be negatively influenced by estrogens used in hormone therapy. OBJECTIVES: We administered two doses of GEN [mean daily uptake 5.4 (low) or 54 mg/kg (high) body weight (bw)] orally over a period of 3 months to ovariectomized (ovx) rats and compared the effects with a treatment with two doses of 17beta-estradiol [E(2); 0.17 (low) or 0.7 mg/kg bw (high)]. Mammary glands, vaginae, and uteri were investigated morphologically and immunohistochemically. We quantified the expression of proliferating cell nuclear antigen (PCNA) and progesterone receptor (PR) in the mammary gland. RESULTS: In rats treated with either of the E(2) doses or the high GEN dose, we found increased uterine weight, and histologic analysis showed estrogen-induced features in the uteri. In vaginae, either E(2) dose or GEN high induced hyperplastic epithelium compared with the atrophic controls. In the mammary gland, E(2) (either dose) or GEN increased proliferation and PR expression. Serum levels of luteinizing hormone were decreased by E(2) (both doses) but not by GEN. CONCLUSIONS: In summary, E(2) and GEN share many effects in the studied organs, particularly in the vagina, uterus, and mammary gland but not in the hypothalamo/pituitary unit.

Endocrine disruptors and the thyroid gland--a combined in vitro and in vivo analysis of potential new biomarkers.

BACKGROUND: There is growing evidence that, in addition to the reproductive system, the hypothalamic-pituitary-thyroid axis is a target of endocrine-disrupting compounds (EDCs). However, this is not reflected adequately in current screening and assessment procedures for endocrine activity that to date determine only general parameters of thyroid function. OBJECTIVE AND METHODS: We used several in vitro and ex vivo assays in an attempt to identify suitable biomarkers for antithyroid action testing a selected panel of putative EDCs. RESULTS: In vitro we detected stimulation or inhibition of iodide uptake into FRTL-5 rat thyroid cells, inhibition of thyroid hormone binding to transthyretin, agonistic or antagonistic effects in a thyroid hormone receptor-dependent reporter assay, and inhibition of thyroid peroxidase using a novel assay system based on human recombinant thyroperoxidase that might be suitable for routine screening for potential EDCs. In rats, chronic application of several EDCs led to changes in thyroid morphology, alterations of thyrotropin and thyroid hormone serum levels as well as alterations in peripheral thyroid hormone-regulated end points such as malic enzyme and type I 5'-deiodinase activity. CONCLUSIONS: As the effects of EDCs do not reflect classic mechanisms of hormone-dependent regulation and feedback, we believe multitarget and multimodal actions of EDCs affect the hypothalamic-pituitary-thyroid axis. These complex effects require a diverse approach for screening, evaluation, and risk assessment of potential antithyroid compounds. This approach involves novel in vitro or cell-based screening assays in order to assess thyroid hormone synthesis, transport, metabolism, and action as well as in vivo assays to measure thyroid hormone-regulated tissue-specific and developmental end points in animals.

Effects of dietary equol on body weight gain, intra-abdominal fat accumulation, plasma lipids, and glucose tolerance in ovariectomized Sprague-Dawley rats.

OBJECTIVE: To evaluate the effects of dietary equol, a metabolite of the phytoestrogen daidzein, on body weight gain, intra-abdominal fat accumulation, plasma leptin, lipids, and glucose tolerance in ovariectomized rats and to compare them to the effects of 17beta-estradiol. DESIGN: Twenty-eight female Sprague-Dawley rats were ovariectomized and fed soy-free chow with the addition of estradiol-3 benzoate (E2B) (10 mg/kg, n=10) or equol (400 mg/kg, n=10). The control group (n=8) received soy-free chow only. Weight and food intake were recorded once weekly. After 6 weeks, intra-abdominal fat was measured using computed tomography, and the intraperitoneal glucose tolerance test was performed. In the seventh week, the animals were killed, blood was collected for plasma, and uteri were removed. RESULTS: Dietary equol significantly increased uterine mass. This effect was, however, 3.5 times lower in magnitude compared to E2B. Similar to E2B, dietary equol decreased weight gain, intra-abdominal fat accumulation, and plasma leptin levels. Equol-treated animals had also lower plasma total cholesterol and triglyceride levels compared to controls. E2B treatment also decreased plasma total cholesterol as well as high-density lipoprotein and low-density lipoprotein cholesterol. In the glucose tolerance test, the area under the curve was significantly smaller in the E2B- and equol-treated animals compared to controls. Also, E2B-treated animals had lower fasting plasma insulin levels. CONCLUSIONS: In ovariectomized rats, dietary equol administration attenuates weight gain and shows favorable metabolic effects. However, because of its mild uterotrophic activity, its use in the prevention of postmenopausal weight gain and related metabolic disorders in women with an intact uterus is questionable in terms of safety and warrants further studies.