Estrogen and raloxifene improve metaphyseal fracture healing in the early phase of osteoporosis. A new fracture-healing model at the tibia in rat.

BACKGROUND: Fracture healing in osteoporosis is delayed. Quality and speed of fracture healing in osteoporotic fractures are crucial with regard to the outcome of patients. The question arises whether established antiosteoporotic drugs can further improve fracture healing. MATERIALS AND METHODS: Osteoporosis manifests predominantly in the metaphyseal bone. Nevertheless, an established metaphyseal fracture model is lacking. A standardized metaphyseal fracture-healing model with stable plate fixation was developed for rat tibiae. The healing process was analyzed by biomechanical, gene expression, and histomorphometric methods in ovariectomized (OVX) and sham-operated rats (SHAM), compared to standardized estrogen (E)- and raloxifene (R)-supplemented diets. RESULTS: Estrogen and raloxifene improved the biomechanical properties of bone healing compared to OVX (Yield load: SHAM = 63.1 +/- 20.8N, E = 60.8 +/- 17.9N, R = 44.7+/-17.5N, OVX = 32:5 +/- 22.0N). Estrogen vs OVX was significant based on a denser trabecular network. Raloxifene greatly induced total callus formation ((R = 5.3 +/- 0.9 mm2, E = 4.7 +/- 0.5 mm2, SHAM = 4.51 +/- 0.61 mm2, OVX =4.1 +/- 0.6 mm2), whereas estrogen mainly enhanced new endosteal bone formation. There was no correlation between the gene expression (osteocalcin, collagen1alpha1, IGF-1, tartrate-resistant phosphatase) in the callus and the morphology and quality of callus formation. CONCLUSION: Raloxifene and estrogen improve fracture healing in osteoporotic bone significantly with regard to callus formation, resistance, and elasticity. The biomechanically stable metaphyseal osteotomy model with T-plate fixation presented here has proven to be appropriate to investigate fracture healing in osteoporosis.

Effects of isoflavones equol and genistein on bone quality in a rat osteopenia model.

Phytoestrogens might be an alternative medication in prophylaxis and treatment of osteoporosis. In this study, the osteoprotective effects of genistein (GEN) and equol (EQO) were evaluated. After ovariectomy, 44 rats received soy-free food (Control, C) and developed substantial osteoporosis over the course of two months. After that period, the rats were divided into different groups and fed estradiol (E), GEN or EQO for 35 days. To analyze the osteoprotective effects of the tested substances, bone biomechanical properties and histomorphometric changes of the lumbar vertebrae were evaluated. In analyzing the vertebral body compression strength, we found that the EQO (103.8%) and GEN (96.8%) groups reached similar levels relative to the E group, while the C group reached 77.7% of the biomechanical properties of the E group. EQO was significantly superior to C. The histomorphometric evaluation demonstrated an increased number of nodes in EQO- and E-treated rats compared to GEN- and C-treated rats. E led to an improvement of cortical as well as trabecular bone, an advantage that was only partly seen in the other groups. Treatment with phytoestrogens induced improved bone quality. EQO and GEN might be alternatives for hormone replacement therapy, although further studies are needed to elucidate possible side effects.

Effect of testosterone, raloxifene and estrogen replacement on the microstructure and biomechanics of metaphyseal osteoporotic bones in orchiectomized male rats.

INTRODUCTION: Currently, osteoporosis research is rarely undertaken in males but an increase in male life expectancy in the company of hypogonadism suggests the necessity for potential therapeutic options. MATERIALS AND METHODS: In this study, the changes in bone structure under standardized testosterone- (T), raloxifene- (R) and estrogen (E)-supplemented diets were analyzed in osteoporotic castrated male rats. RESULTS: Unexpected biomechanical results could be only explained by the histomorphometry, but not by BMD measurements obtained from the qCT. All tested substances showed a significant improvement in the trabecular network (trabecular bone area for C: 2.55 mm(2), T: 4.25 mm(2), R: 4.22 mm(2) and E: 4.28 mm(2)), and suggests that the bone structure was preserved. For the metaphyseal cortical bone, a significant loss was detected in T (CBP: 18.7%) compared to R (CBP: 30.0%), E (CBP: 26.8%) and even to the osteoporotic control (CBP: 28.6%). This explains the observed early mechanical final failure after T supplementation. However, due to the preserved trabecular bone in T, the occurrence of the first microfractures (yL: 49 +/- 21.4 N) was significantly later than in the osteoporotic control (yL: 39.5 +/- 15.5 N). Raloxifene performed well in hindering the bone loss associated with osteoporosis. However, its effect (yL: 83.3 +/- 16.5 N) did not approach the protective effect of E (yL: 99.2 +/- 21.1 N). CONCLUSION: Testosterone only preserved the deterioration of the trabecular bone but not of the cortical bone. Raloxifene prevented the bone loss associated with osteoporosis at all bony structures. This effect did not approach the protective effect of estrogen on trabecular bone, but it is more suitable for male individuals because it has no feminizing effects on the subject.

Vitex agnus castus as prophylaxis for osteopenia after orchidectomy in rats compared with estradiol and testosterone supplementation.

Osteoporosis research undertaken in males is rare and there are only a few therapeutic options. Phytoestrogens might be a safe alternative for prophylaxis. Sixty 3-month-old male rats were orchidectomized and divided into five groups. The groups either received soy-free food (C), estradiol (E), testosterone (T) or Vitex agnus castus in different concentrations (AC high/AC low) for 12 weeks. The tibia metaphysis was tested biomechanically and histomorphometrically. The AC high group reached 87% of the biomechanical values of the estradiol group and was significantly superior to the control group. Testosterone supplementation resulted in poor biomechanical properties. The cortical bone parameters of the AC group were similar to the control group, while supplementation with estradiol and testosterone demonstrated a reduction of cortical bone. The AC high group reached 88.4% of trabecular bone area, 80.7% of trabecular number and 66.9% of the number of trabecular nodes compared with estradiol supplementation. Vitex agnus castus demonstrated osteoprotective effects in males. It preserves the cortical as well as the trabecular bone and might be a safe alternative for HRT. Testosterone supplementation has positive effects on trabecular bone, which are concurrently counteracted by the loss of cortical bone.

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.

Evaluation of the antiosteoporotic potential of Tinospora cordifolia in female rats.

UNLABELLED: The available courses of therapy to osteoporosis in menopausal women are limited by several side effects generated. A need therefore arises to explore herbal alternatives that are effective and safe. OBJECTIVE: Present animal studies were conducted to investigate the potential of Tinospora cordifolia (TC) ethanolic stem extract as an antiosteoporotic agent. METHODS: Three-month-old female Sprague-Dawley rats were either ovariectomized (ovx) or sham operated and treated with vehicle (benzyl benzoate:castor oil; 1:4), E(2) (1 microg/day) or TC (10, 50, 100 mg/kg b.wt) subcutaneously for 4 weeks. At the end of experiment bone mineral density of tibiae was measured by quantitative computer tomography. Serum was analyzed for the activity of alkaline phosphatase and levels of osteocalcin, cross-laps and lipids. Uterus and mammary gland were processed for histological studies. RESULTS: Ovx rats treated with TC (10 mg/kg b.wt) showed an osteoprotective effect as the bone loss in tibiae was slower than ovx controls. Serum osteocalcin and cross-laps levels were significantly reduced. All the above effects of TC were much milder than those produced by E(2). Alkaline phosphatase activity was higher in TC treatment groups. Total cholesterol and LDL levels remained unaltered but HDL levels were significantly lowered with TC (50 mg/kg b.wt) treatment. Uterus and mammary gland showed no signs of proliferation after treatment with TC extract. CONCLUSION: TC extract showed estrogen like effects in bone but not in reproductive organs like uterus and mammary gland. Thus, this study demonstrates that extract of T. cordifolia has the potential for being used as antiosteoporotic agent.

[Pharmacological potential of phytoestrogens in the treatment of prostate cancer]. / Das pharmakologische Potential von Phytoöstrogenen in der Therapie des Prostatakarzinoms.

INTRODUCTION: Phytoestrogenes are plant-derived compounds that have been shown to exert an antiproliferative potential on prostate cancer cells, although the exact mechanisms are still unclear. In prostate cancer cells proliferation is regulated by modulation of the IGF-1 receptor (IGF-R-1) by the androgen receptor (AR) and its co-activator prostate derived Ets factor (PDEF). Phytooestrogenes interact with these mechanisms as demonstrated exemplarily in the presented study with the isoflavone tectorigenin derived from Belamcanda chinensis. MATERIAL AND METHODS: Cultured androgen-sensitive LNCaP prostate cancer cells were treated with tectorigenin of 100 microM for 24 hours. The mRNA-expression of AR, PSA, PDEF, hTERT, TIMP-3 and IGF-R-1 were quantified by real-time RT-PCR. Furthermore, the expression or activity of PSA, telomerase and IGF-R-1 was measured on the protein level. In addition, we investigated in nude mice the influence of a diet of extracts of Belamcanda chinensis on the growth of subcutaneously injected LNCaP cells versus a control group of animals fed with a soy-free diet. RESULTS: In cultured LNCaP cells treatment with tectorigenin resulted in a significant down-regulation of the gene expression of AR, PDEF, PSA, IGF-R-1 and hTERT. On the protein level PSA secretion and the activity of telomerase and IGF-R-1 expression was also decreased. The gene expression of TIMP-3 was distinctly up-regulated by tectorigenin. Nude mice fed with Belamcanda chinensis extract showed a significantly decreased incidence and tumor growth compared to controls. CONCLUSIONS: Tectorigenin shows an inhibition of the IGF-1-R modulated cell proliferation of PCa-Cells, due to modulation of the activity the co-activator PDEF independently from the AR. Furthermore, tectorigenin has pro-apoptotic effects and decreases tissue invasion by up-regulation of TIMP-3. Therefore, phytooestrogenes are an interesting option in the therapy of prostate especially advanced prostate cancer.

Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis.

Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17alpha (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary-gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.

Effects of estradiol-17beta, testosterone and a black cohosh preparation on bone and prostate in orchidectomized rats.

Estradiol (E2) and testosterone (T) effectively prevent orchidectomy (orx) induced osteoporosis. T, however, stimulates prostate proliferation which may lead to malignancy. We showed that a Cimicifuga racemosa (CR) preparation had bone-sparing effects without exerting estrogenic effects in the uterus. We studied therefore whether a CR preparation has also antiosteoporotic effects in orx rats substituted with E2, T or CR via pelleted food over a period of 3 months. Average daily intake per animal was: T: 25 mg; E2: 0.325 mg, CR low dose: 33 mg; CR high dose: 133 mg. E2, T and CR at the high dose partially prevented development of osteoporosis as measured by quantitative computer tomography in the metaphysis of the tibia. E2, but not T or CR reduced serum osteocalcin and the metabolic products of collagen-1alpha1. Gene expression of collagen-1alpha1 and tartrate-resistant acid phosphatase was decreased by E2 and the higher dose of the CR extract but increased in the T-treated animals. In the prostate T inhibited androgen receptor, estrogen receptor alpha and insulin-like growth factor-1 gene expression but stimulated the expression of the ERbeta gene. These effects were not shared by E2 or both doses of the CR extract. It is concluded that E2, T and CR exert antiosteoporotic effects in the metaphysis of the tibia of orx rats. T has profound effects in the prostate which were not seen in the E2- and CR-treated animals. Therefore, the Cimicifuga racemosa extract BNO 1055 may be useful to prevent osteoporosis in aged male patients with reduced testosterone production.

Silymarin is a selective estrogen receptor beta (ERbeta) agonist and has estrogenic effects in the metaphysis of the femur but no or antiestrogenic effects in the uterus of ovariectomized (ovx) rats.

Silymarin is a widely used standardized mixture of flavonolignans and its major component Silybinin binds to cytosolic estrogen receptors. Here, we demonstrate that this binding is exclusive to the estrogen receptor beta (ERbeta). Treatment of ovariectomized (ovx) rats with silymarin or estradiol (E2) may allow differentiation of biological effects mediated by the ERalpha or ERbeta. E2 inhibited serum LH, cholesterol, LDL and HDL concentrations in the blood and increased gene expression of IGF1, HbEGF and C3 in the uterus, while silymarin was totally ineffective or antagonistic in altering these parameters. Both, E2 and silymarin inhibited expression of uterine ERbeta gene. Hence, in the pituitary, liver (where the lipoproteins are synthesized) and uterus E2 acts primarily via the ERalpha. Exclusive estrogenic effects of silymarin were observed in the metaphysis of the femur (MF), on osteoblast parameters (gene expression of IGF1, TGFbeta1, osteoprotegerin, collagen-1alpha1, osteocalcin (OC)) and on the osteoclast activity marker tartrate resistant acid phosphatase (TRAP) gene expression of adult ovx rats. Our RT-PCR method detects ERbeta gene expression in all organs including developing bones but not in the MF of adult ovx rats. We conclude therefore, that the effects of silymarin in this part of the bone cannot be exerted via the ERalpha because it does not bind to this receptor subtype. Despite the failure to detect ERbeta mRNA in the MF of our animals the possibility exists that ERbeta protein is present and may mediate the effects of silymarin. Another possibility may be that the effect of silymarin and therefore possibly also of E2 in the MF may be mediated via other possibly not yet identified receptors or via an ERbeta splice variant which is not detected by our PCR-method.

Phytoestrogens for hormone replacement therapy?

Due to some severe side effects "classical" hormone replacement therapy (HRT) is currently being challenged by a therapy with phytoestrogens. Particularly soy and red clover derived isoflavones are advertised as selective estrogen receptor modulators (SERMs) with only desired and no undesired estrogenic effects. Evidence that this is the case however is scarce. Most studies investigating climacteric complaints did not find beneficial effects. A proposed beneficial effect on mammary cancer is unproven. The majority of studies however indicate an antiosteoporotic effect of isoflavones, while putative beneficial effects in the cardiovascular system are questionable due to the fact that estradiol which--like isoflavones--increase HDL and decrease LDL concentrations appear not to prevent arteriosclerosis in the human. In the urogenital tract, including the vagina, soy and red clover derived isoflavones are without effects. Cimicifuga racemosa extracts are traditionally used for the treatment of climacteric complaints. Evidence is now available that the yet unknown compounds in Cimicifuga racemosa extracts prevent climacteric complaints and may also have antiosteoporotic effects.

Pure estrogenic effect of benzophenone-2 (BP2) but not of bisphenol A (BPA) and dibutylphtalate (DBP) in uterus, vagina and bone.

UNLABELLED: Contradictory results whether the endocrine disrupters (ED) benzophenone-2 (BP2), bisphenol A (BPA) and dibutylphtalate (DBP) exert estrogenic effects have been published. Selective estrogen receptor modulators (SERMs) exert estrogenic effects in some but not in all organs and ED may be SERMs. Therefore, we studied their binding properties to recombinant ERalpha and ERbeta protein and their effects in the uterus, vagina and bone of ovariectomized rats. BP2 bound to both receptor subtypes, while BPA had a relatively high ERbeta selectivity. DBP did not bind to ERalpha but with a low affinity to ERbeta. In the uterus, only E2 and BP2 increased uterine weight and the complement C3 but decreased ERbeta gene expression. Discrete effects of BPA and DBP in the uterus were found upon histological examination. In the vagina, BP2 but not BPA and DBP had clear estrogenic effects. E2 and BP2 had antiosteoporotic effects in the metaphysis of the tibia. The serum surrogate parameters of bone metabolism, i.e. osteocalcin and the cross (rat) laps were significantly reduced by E2, an effect shared with BP2 but not by the two other EDs. THE CONCLUSION: BP2 acts as ERalpha and ERbeta agonist mimicking effects of E2, while the effects of BPA and DBP are not pure estrogenic.

In vivo properties of the urinary bladder wall and their modulation by estradiol and raloxifene in a rat model.

OBJECTIVES: Urinary incontinence is a common symptom of urogenital aging that affects a considerable proportion of postmenopausal women. Morphological and morphometrical modulation of the bladder by estrogen are known. Yet data showing that this translates into changes of in vivo function of the urinary bladder are missing. METHODS: We measured urodynamic parameters in anaesthetized, surviving rats. Following ovariectomy animals were divided into three groups and fed either an estradiol-, raloxifene-, or unsupplemented soy-free formula for ten weeks. Via a transurethral catheter the intravesical pressure was recorded during a stretch period (the urinary bladder was filled), and a one-minute isometric accommodation period immediately after the filling period. Upon termination of the experiment upper and lower halves of the bladder were processed histologically. RESULTS: The estrogen-, and raloxifene-treated animals showed significantly higher pressures in responses to rapid stretch. Bladder compliance during the isometric period on the other hand was not significantly affected by these treatments. Thickness of the epithelial layer, collagen content and muscle bundles were significantly increased by estrogen and raloxifene treatment. CONCLUSIONS: This is a good animal model to investigate modulation of detrusor muscle contractility and stiffness. Both estradiol and raloxifene increase bladder contractility. Urinary bladder morphology indicates that estrogen acts primarily in the upper half of this organ since significant effects on collagen content and muscle fibers are only found in this part.

The Cimicifuga preparation BNO 1055 vs. conjugated estrogens in a double-blind placebo-controlled study: effects on menopause symptoms and bone markers.

OBJECTIVES: In the present study, therapeutic effects of the Cimicifuga racemosa preparation CR BNO 1055 (Klimadynon/Menofem) on climacteric complaints, bone metabolism and endometrium will be compared with those of conjugated estrogens (CE) and placebo. The question whether CR BNO 1055 contains substances with selective estrogen receptor modulator (SERM) activity will be investigated. METHODS: Sixty-two evaluable postmenopausal women were included in the double-blind, randomized, multicentre study, and treated either with CR BNO 1055 (daily dose corresponding to 40 mg herbal drug), 0.6 mg CE, or matching placebo, for 3 months. Menopausal symptoms were assessed by the menopause rating scale (MRS) and a diary. Levels of CrossLaps (marker of bone degradation) were determined by ELECSYS system and bone-specific alkaline phosphatase (marker of bone formation) by an enzymatic assay. Endometrial thickness was measured via transvaginal ultrasound; vaginal cytology was also studied. The primary efficacy criterion was the change from baseline to end point in the MRS. Change from baseline was analyzed for the secondary variables too. RESULTS: CR BNO 1055 proved to be equipotent to CE and superior to placebo in reducing climacteric complaints. Under both verum preparations, beneficial effects on bone metabolism have been observed in the serum. CR BNO 1055 had no effect on endometrial thickness, which was significantly increased by CE. Vaginal superficial cells were increased under CE and CR BNO 1055 treatment. CONCLUSION: The results concerning climacteric complaints and on bone metabolism indicate an equipotent effect of CR BNO 1055 in comparison to 0.6 mg CE per day. It is proposed that CR BNO 1055 contains substances with SERM activity, i.e. with desired effects in the brain/hypothalamus, in the bone and in the vagina, but without exerting uterotrophic effects.

Pharmacology of Cimicifuga racemosa extract BNO 1055 in rats: bone, fat and uterus.

OBJECTIVES: Hormone replacement therapy (HRT) has therapeutic effects on climacteric complaints and prevents osteoporosis. Owing to the increased risks of breast cancer and cardiovascular diseases, patients look for alternatives. Cimicifuga racemosa (CR) preparations might be an alternative, because they proved to reduce climacteric complaints as efficiently as conjugated estrogens without exerting estrogenic effects in the uterus. Whether CR has positive effects on bone and in fat tissue is currently unknown. Therefore, osteoprotective effects of the CR extract BNO 1055 and an influence on fat tissue were studied in ovariectomized rats. METHODS: Bone mineral density (BMD) of the tibia of ovariectomized (ovx) rats was determined by computer-assisted tomography (CT). CT scans of fat depots were perimetrically quantified. Bone turnover (osteocalcin, crosslaps) and lipocyte activity (leptin) were also determined. Uterine weights were measured and gene expression of estrogen-regulated uterine genes (IGF-1, ERbeta) was determined by RT-PCR. RESULTS: Treatment of the ovx rats over a period of 3 months with E(2) and the CR extract BNO 1055 showed osteoprotective effects; both significantly reduced the loss of BMD in tibia. Serum osteocalcin levels were significantly reduced by both treatments, whereas only E(2), but not BNO 1055, reduced serum crosslaps. A paratibial fat depot and serum leptin concentration were also significantly reduced. In contrast to E(2), the CR extract showed no effect on uterine weight and gene expression of E(2)-regulated genes. CONCLUSION: The CR extract BNO 1055 exerted estrogenic effects in the bone (particularly in osteoblasts) and in fat tissue, but not in the uterus of ovx rats. The extract appears to contain rat organ-specific selective estrogen receptor modulators (SERMs), and if these findings can be approved in human it may be an alternative to HRT.

Beneficial effects of beta-Ecdysone on the joint, epiphyseal cartilage tissue and trabecular bone in ovariectomized rats.

Ecdysteroids are steroids found in invertebrates and plants. In mammals they have protein anabolic effects. We have recently published antiosteoporotic effects of Tinospora cordifolia (TC) extract and the search for the possible active ingredients yielded the presence of beta-Ecdysone (Ecd). Therefore, we investigated the effects of pure Ecd in ovariectomized rats on morphological changes in joint, epiphyseal cartilage and trabecular tissue. Following ovariectomy rats were fed for 1 month with Ecd containing food at a dose of 52.8 mg/day/animal. Positive and negative control animals received 17-beta Estradiol (E(2), 132 microg/day/animal) and soy free (sf) food respectively. At sacrifice, specimens consisting of upper tibiae-lower femurs and knee joint were harvested and processed for histomorphometry. The parameters measured included thickness of the joint cartilage, thickness of the whole epiphyseal growth plate and its three zones. Furthermore, the percentage of trabecular bone in the metaphysis region of tibiae was quantified. Ecd and E(2) induced a significant increase in the thickness of joint cartilage. The whole epiphyseal growth plate and its proliferative and hypertrophic zones were also increased by Ecd whereas E(2) reduced their size. The percentage of trabecular area in the metaphysis of tibia was significantly increased in Ecd and E(2) treated animals. Results provide a plausible explanation for the antiosteoporotic effects of TC. Hence, TC as well as other Ecd producing plants or pure Ecd may be of value in the prevention and treatment of osteoporosis and osteoarthritis which is of increasing importance due to aging and obesity among individuals.

Effects of dietary equol on the pituitary of the ovariectomized rats.

The aim of our study was to evaluate the effects of dietary equol, metabolite of a phytoestrogen daidzein, on the secretion of prolactin (PRL) and lutenizing hormone (LH), as well as the expression of estrogen receptors (ERalpha, ERbeta and truncated estrogen receptor-1 (TERP-1) in the pituitary gland of ovariectomized (ovx) female Sprague-Dawley rats. Two doses of equol (50 mg/kg of chow and 400 mg/kg of chow) were used and the results were compared with the effects of estradiol 3-benzoate (E2B), also given at two doses (4.3 mg/kg of chow and 17.3 mg/kg of chow). Treatment period was 3 months. Dietary equol administration at the high dose increased significantly serum PRL levels. This effect was also observed in the E2B group but this difference did not reach statistical significance. Surprisingly, high dose dietary equol treatment also significantly increased serum LH levels, which was in contrast to E2B treatment where serum LH levels were significantly decreased at both doses. Serum LH levels in the equol low group were unaffected. Equol treatment had no effects on pituitary ERalpha or ERbeta gene expression. In contrast, high dose E2B treatment increased significantly pituitary ERalpha mRNA levels but decreased those of ERbeta. Both doses of E2B also increased significantly pituitary TERP-1 mRNA levels. This effect was also observed in the equol high group but at a much smaller magnitude. In conclusion, high dose dietary equol administration to ovx rats exerts estrogenic like effects on the lactotropes and anti-estrogenic on the gonadotropes.

Uterotropic effects of dietary equol administration in ovariectomized Sprague-Dawley rats.

AIM: The aim of the present study was to evaluate the uterotropic effects of the administration of dietary equol, a metabolite of soy-derived daidzein or formononetin present in red clover, in an ovariectomized rat model of menopause. METHOD: Two doses of racemic equol were used (50 mg/kg of chow and 400 mg/kg of chow) and the results were compared with two doses of estradiol-3 benzoate (E2B) (4.3 mg/kg of chow and 17.3 mg/kg of chow). After 3 months, animals were sacrificed and the uteri were removed, weighed and paraffin-embedded for morphometrical and immunohistochemical evaluation. The expression of selected uterine estrogen-responsive genes was also measured using real-time reverse transcription-polymerase chain reaction. RESULTS: Compared to controls, uterine weights in animals treated with high-dose equol were significantly higher, presented histologic features of mild estrogenic stimulation and had greater epithelial height and thickness of the uterine stroma and myometrium. Staining for the presence of the proliferating cell nuclear antigen (PCNA) also showed a greater prevalence of the PCNA-positive cells in the uterine stroma in animals treated with high-dose equol. Conversely, the percentage of PCNA-positive cells in the uterine epithelium was lower compared to the controls. Dietary high-dose equol treatment also increased significantly levels of uterine insulin-like growth factor 1, progesterone receptor and complement protein 3 mRNA. Although statistically significant, all these effects were, however, lower in magnitude compared to the effects of low- and high-dose E2B treatment. Low-dose equol did not have any effects on the above-studied parameters. CONCLUSION: Long-term high-dose dietary equol administration to ovariectomized rats exerts uterotropic effects at the cellular and molecular level which question the safety of uncontrolled and unlimited consumption of soy or red clover supplements by postmenopausal women with intact uteri.

Comparison of effects of estradiol (E2) with those of octylmethoxycinnamate (OMC) and 4-methylbenzylidene camphor (4MBC)--2 filters of UV light - on several uterine, vaginal and bone parameters.

OMC and 4MBC are 2 absorbers of ultraviolet light which are used in unknown quantities in sunscreens, cosmetics and plastic products to protect against UV light-induced damage of the skin or of fragrances or plastic material. From there, they were shown to reach surface water and/or by direct contamination or ingestion the human. Under various conditions in mice and rats, both substances were shown to be estrogenic. Therefore, we compared in vitro and in vivo the effects of chronic application of these compounds at 2 doses with those of E2, all administered via food. No signs of toxicity were observed under application of 0.6 mg E2, 57.5 or 275 mg of OMC, 57.5 or 250 mg of 4MBC; these amounts were ingested with 21 g of control food, 17.8 g E2 food, 20.6 g or 22.3 g OMC food and 23.7 or 22.8 g 4MBC food. In the uterus, vagina and bone, E2 exerted the expected stimulatory effects which were minimally shared by OMC and 4MBC in the uterus and vagina as assessed by histology and determination of a variety of estrogen-regulated genes such as insulin-like growth factor-1, progesterone receptor and estrogen receptor beta. In the bone, OMC had no effect, while 4MBC shared the antiosteoporotic effects of E2 as measured by quantitative computer tomography in the metaphysis of the tibia. The mechanism of action of 4MBC, however, appears to be different as E2 reduced serum osteocalcin and the C-terminal breakdown products of collagen-1alpha1 which were both increased by 4MBC. Taken together, these data indicate a very weak estrogenic effect of OMC and 4MBC in the uterus and in the vagina but not in the bone where 4MBC exerted antiosteoporotic effects by a different mechanism than E2.

Phytoestrogen resveratrol suppresses steroidogenesis by rat adrenocortical cells by inhibiting cytochrome P450 c21-hydroxylase.

BACKGROUND AND AIM: The phytoestrogen resveratrol is found in grapes, mulberries and peanuts, all of which are consumed regularly by humans. Resveratrol is also used in chemotherapy against cancer and aging and as a cardioprotectant. The aim of the present study was to characterize the effects of resveratrol on rat adrenal steroidogenesis and to study the underlying mechanism. METHODS: Adrenocortical cells were isolated from the adrenal glands of normal male rats (in vitro) and from male rats administered resveratrol in their diet for 12 weeks (ex vivo). Cells from resveratrol-treated and non-treated rats were tested ex vivo for responsiveness to ACTH and cells from normal rats were tested in vitro for responsiveness to ACTH in the presence and absence of resveratrol. Corticosterone and progesterone production were measured by RIA and expression of steroidogenic enzymes analyzed by PAGE/Western blotting. RESULTS: Corticosterone production was inhibited 47% by 50 microM resveratrol in vitro and 20% ex vivo, while progesterone production was elevated to 400% of the control value in in vitro experiments. Resveratrol treatment decreased adrenal cytochrome P450 c21-hydroxylase expression in vivo and cell culture conditions. No changes in cell viability or morphology were caused by exposure to resveratrol in both ex vivo and in vitro experiments. CONCLUSION: Resveratrol suppresses corticosterone production by primary rat adrenocortical cell cultures in vitro and ex vivo by inhibiting cytochrome P450 c21-hydroxylase.