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.

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.

Effects of black cohosh (Cimicifuga racemosa) and estrogen on metaphyseal fracture healing in the early stage of osteoporosis in ovariectomized rats.

Osteoporosis and its accompanying, predominantly metaphyseal, fractures are a major health problem. Black cohosh (Cimicifuga racemosa) and estrogen positively influence osteoporotic bone. Both substances may improve fracture healing in early osteoporosis as well. In 48 twelve-week-old ovariectomized or, respectively, sham-operated (SHAM) rats, a standardized metaphyseal tibia osteotomy with bridging T-plate fixation was performed. During the healing process of 35 days, rats received soy-free (SHAM, osteopenic C), estrogen- (E) or Cimicifuga racemosa- (CR) supplemented diets. After sacrifice, the callus formation was analyzed with regard to biomechanical quality, morphology, quantity, time course of new bone built and gene expression. CR induced a high rate of metaphyseal callus formation. The biomechanical properties and the amount of new callus formation indicated that fracture healing was still in progress. Therefore, gene expression of osteoblasts was comparatively high. Body weight and the trabecular structure were influenced little by CR. Estrogen improved the biomechanical properties of the callus. Resistance to microfracturing was significantly enhanced in the E group and even superior to SHAM. Remodeling of the callus formation had already begun. The trabecular network and the typical endosteal fracture healing were especially improved. Osteoporotic metaphyseal fracture healing was improved by estrogen more than by Cimicifuga racemosa. The process of fracture healing occurred nearly physiologically. The generation of callus formation was supported by Cimicifuga racemosa as well, but the five-week duration of application was too short for Cimicifuga racemosa to show its complete potential. Already-initiated Cimicifuga racemosa therapy for menopausal symptoms could be continued during fracture healing without hesitation.

Effect of dihydrotestosterone, 17-ß-estrogen, genistein and equol on remodeling and morphology of bone in osteoporotic male rats during bone healing.

INTRODUCTION: The aim of this study was to investigate the effect of dihydrotestosterone (DHT), 17-ß-estrogen (E2), genistein (GEN) and equol (EQ) on bone remodeling and bone morphology during healing of osteoporotic male rat tibiae. MATERIALS AND METHODS: 180 Sprague-Dawley male rats were divided in 5 groups of 36 animals. After orchidectomy (ORX) and development of osteoporosis, trepanation of the tibia was performed. Until the time of trepanation all groups received soya free food (SF), then food change occurred and treatment started. At day 95, 102 and 151, samples were taken and histomorphometry was performed to analyze changes in bone structure under treatment. At day 33 and 70 all animals received calcein respective alizarin for polychrome bone labeling. RESULTS: The cortical bone was particularly affected. Treatment with DHT and E2 led to a significant long-term expansion of the thickness of the diaphyseal cortical bone, while the phytoestrogens EQ and GEN only had a positive short-term effect in this area. Only E2 preserved the trabecular bone for a limited time. In all groups, periosteal and endosteal bone areas showed the highest bone formation activity. The osteoporotic male injured bone shows a shift in mineral apposition rate (MAR) from periosteal to endosteal bone in the SF, DHT and E2 groups but not in the GEN and EQ phytohormones groups. An MAR decrease in trabecular bone formation was observed at day 70 in all groups except the E2 group. CONCLUSION: We conclude from our results that healing of cortical bone defects in a rat model of male osteoporosis are mainly influenced by the estrogen pathway. Nevertheless, effects via purely androgenic mechanisms can also be demonstrated. The role of a phytohormone therapy is only marginal and if only useful for a short-term supportive approach. The role of the periosteal to endosteal shift during male osteoporotic bone healing needs to be further examined.

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.

Effect of ovariectomy on proximal tibia metaphysis and lumbar vertebral body in common marmoset monkeys.

This study aimed to investigate the effect of estrogen withdrawal on bone tissue in adult female marmoset monkeys. In a 1-year follow-up study we used quantitative computer tomography to measure total bone mineral density (BMD) of the proximal tibia and the second-last lumbar vertebral body (L5/L6) before and 1, 3, 6, and 12 months after ovariectomy. Body mass did not significantly change during the 1-year observation period. However, a significant decline of total BMD after ovariectomy was observed in the proximal tibia but not in L5/L6. In addition, regression analysis showed a significant positive relationship between BMD and body mass in both tibia and L5/L6. The results of our study support the idea that ovariectomized marmoset monkeys may serve as a model to investigate bone loss related to decline of estrogen production.

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 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.

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.

Effects of dietary equol administration on ovariectomy induced bone loss in Sprague-Dawley rats.

UNLABELLED: Oestrogen deficiency leads to a considerable bone loss, thus, osteopenia and osteoporosis are serious complications after menopause. OBJECTIVES: To evaluate the effects of a daidzein metabolite equol on bone mass density (BMD) and markers of bone remodelling in an ovariectomized (ovx) rat model of postmenopausal bone loss and compare them with the effects of 17beta-estradiol. METHODS: Twenty-eight female Sprague-Dawley rats were ovx and fed soy-free chow only (control group, n = 8), or with the addition of oestradiol-3 benzoate (E2B) (10mg/kg, n = 10) or equol (400 mg/kg, n = 10). At baseline and after 6-week treatment period, proximal tibia and lumbar spine BMD were measured using computer tomography. Animals were then sacrificed, blood was collected and uteri were removed. RESULTS: Similarly to E2B, dietary equol decreased weight gain and showed mild uterotropic activity. E2B attenuated ovx induced BMD loss at proximal tibia whereas equol had no effect. At lumbar spine, however, equol not only attenuated trabecular bone loss but also increased its density. This effect was also apparent in animals treated with E2B. Cortical BMD at proximal tibia and lumbar spine were not very much influenced by ovx and treatment with E2B or equol did not induce significant changes at these sites. Plasma osteocalcin and type I collagen fragments (cross-laps) in equol treated animals did not differ from the controls whereas in E2B treated animals they were both significantly decreased. CONCLUSIONS: In spite of its mild uterotropic potential, dietary equol shows limited bone sparing effects in ovx rats.

Dietary daidzein and puerarin do not affect pituitary LH expression but exert uterotropic effects in ovariectomized rats.

OBJECTIVE: To investigate the potency of LH suppression, as an indirect measure of alleviation of postmenopausal vasomotor symptoms, as well as the uterotropic effects of two isoflavones: daidzein and puerarin in an ovariectomized (ovx) rat model and compare them with the effects of 17beta-estradiol benzoate (E2B). DESIGN: Eighty female Sprague-Dawley rats were ovx and divided into six different treatment groups and one control group (11-12 animals per group). Daidzein, puerarin and E2B were added to the soy free rodent chow in low and high doses (250 mg and 1000 mg per kg, 600 mg and 3000 mg per kg and 4.3 mg and 17.3 mg per kg, respectively). After 3 months of treatment, animals were sacrificed and using real time RT-PCR, pituitary LHbeta and uterine IGF-1, PR and C3 mRNA levels were measured. Additionally serum LH levels were measured in a radioimmunoassay. RESULTS: Both of our tested isoflavones at low and high doses had no effect on the expression of the pituitary LH at the mRNA and protein level. Only E2B at both doses significantly decreased pituitary LHbeta gene expression and serum LH levels. Daidzein and puerarin at high dose increased significantly uterine weights. Uterine IGF-1 gene expression was only upregulated in puerarin high group. Uterine PR mRNA levels were higher in animals fed with low dose daidzein and high dose puerarin. Uterine C3 gene expression was upregulated in animals fed with daidzein and puerarin at high doses. Although statistically significant, all these effects were however very discrete compared to those of E2B at low and high doses. CONCLUSION: We speculate that due to the lack of LH suppressing effects in our model, it is very unlikely for daidzein and puerarin to alleviate vasomotor symptoms in postmenopausal women. In contrast, due to their uterotropic effects, high dose consumption of commercially available preparations containing daidzein or puerarin may expose women with an intact uterus to the risk of endometrial hyperplasia.

Standardized bending and breaking test for the normal and osteoporotic metaphyseal tibias of the rat: effect of estradiol, testosterone, and raloxifene.

UNLABELLED: The fracture of bone plays a key role in osteoporosis. BMD measurement, however, is only an indirect parameter of this phenomenon. We therefore developed a highly sensitive three-point bending test for the metaphyseal tibias in rats to evaluate stiffness and strength. This was validated in a right-left comparison and a bioassay with soy-free food, estradiol, raloxifene, and testosterone in orchidectomized rats. INTRODUCTION: Osteoporosis becomes manifest predominantly in the metaphyseal rat tibia. The anti-osteoporotic character of substances should, therefore, be tested (mechanically) in this bone area. MATERIALS AND METHODS: We evaluated a new three-point bending test for the metaphyseal tibia in rats in a right-left trial. In an animal experiment, we studied the change of bone quality under estradiol (E)-, raloxifene (R)-, and testosterone (T)-supplemented food and compared it with trabecular BMD (qCT). RESULTS: In the right-left comparison, the mean difference between the metaphyseal loads of both tibias in 37 rats was 8.43% for the maximum load (Fmax) and 6.46% for the failure load (fL). These results show the high reproducibility of the test, because they are close to the usual intraindividual difference of the two extremities. In a second experiment, four groups of 11 3-month-old male orchidectomized rats were fed with soy-free food only (C) or with the additives E, T, or R for 12 weeks. E and R were similar for Fmax and fL. There were significant differences in the stiffness (E = 406.92 N/mm versus R = 332.08 N/mm), the yield load (yL; E = 99.17 N versus R = 83.33 N), and the ratio between yL and Fmax (E = 86.33% versus R = 76.37%). T was similar to the controls concerning F(max), fL, and stiffness. There were significant differences in yL (T = 49.00N versus C = 39.5N) and the ratio between yL and Fmax (T = 64.28% versus C = 51.28%). CONCLUSIONS: Estradiol is superior to raloxifene concerning stiffness and yield load, and both are superior to testosterone. We conclude that the described three-point bending test for the metaphyseal tibia is a highly sensitive method to study hormones and substances with regard to their osteoprotective character. The precision and the low SD of the presented results are superior to the data from qCT and the calculated index of stiffness (SSI).

Effects of black cohosh (Cimicifuga racemosa) on bone turnover, vaginal mucosa, and various blood parameters in postmenopausal women: a double-blind, placebo-controlled, and conjugated estrogens-controlled study.

OBJECTIVES: In this study, the effects of the Cimicifuga racemosa (CR) preparation CR BNO 1055 on markers of bone metabolism, hormones, sex hormone-binding globulin (SHBG), lipometabolism, vaginal maturity, and routine laboratory parameters were compared with those of conjugated estrogens (CE) and placebo. DESIGN: Sixty-two postmenopausal women were included in this double-blind study. Treatment duration with CR (daily dose corresponds to 40 mg of herbal drug), CE (0.6 mg/day), or placebo was 12 weeks. Markers of bone turnover (bone-specific alkaline phosphatase, CrossLaps), estradiol, follicle-stimulating hormone, leuteinizing hormone, SHBG, triglycerides, total cholesterol, high-density cholesterol, low-density cholesterol, and routine clinical chemistry parameters were determined from blood samples. Vaginal "maturity index" was determined from vaginal smears. RESULTS: The analyses of bone turnover markers indicated beneficial effects for CR and CE on bone metabolism. CR stimulated osteoblast activity, whereas CE inhibited osteoclast activity. Whereas CE showed strong estrogenic effects on vaginal mucosa, CR showed weak estrogen-like activity. No significant effects were seen on coagulation markers and liver enzymes in the blood. CR was well tolerated. CONCLUSION: These results suggest that CR has beneficial bone remodeling and weak estrogen-like effects in the vaginal mucosa.