phytoestrogens and estradiol-3-benzoate

The organization of the developing male rodent brain is profoundly influenced by endogenous steroids, most notably estrogen. This process may be disrupted by estrogenic endocrine disrupting compounds (EDCs) resulting in altered sex behavior and the capacity to attract a mate in adulthood. To better understand the relative role each estrogen receptor (ER) subtype (ERα and ERβ) plays in mediating these effects, we exposed male Long Evans rats to estradiol benzoate (EB, 10 μg), vehicle, or agonists specific for ERβ (DPN, 1 mg/kg) or ERα (PPT, 1 mg/kg) daily for the first four days of life, and then assessed adult male reproductive behavior and attractiveness via a partner preference paradigm. DPN had a greater adverse impact than PPT on reproductive behavior, suggesting a functional role for ERβ in the organization of these male-specific behaviors. Therefore the impact of neonatal ERβ agonism was further investigated by repeating the experiment using vehicle, EB and additional DPN doses (0.5 mg/kg, 1 mg/kg, and 2 mg/kg bw). Exposure to DPN suppressed male reproductive behavior and attractiveness in a dose dependent manner. Finally, males were exposed to EB or an environmentally relevant dose of genistein (GEN, 10 mg/kg), a naturally occurring xenoestrogen, which has a higher relative binding affinity for ERβ than ERα. Sexual performance was impaired by GEN but not attractiveness. In addition to suppressing reproductive behavior and attractiveness, EB exposure significantly lowered the testis to body weight ratio, and circulating testosterone levels. DPN and GEN exposure only impaired behavior, suggesting that disrupted androgen secretion does not underlie the impairment.

Topics: Animals; Dose-Response Relationship, Drug; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Genistein; Male; Mating Preference, Animal; Nitriles; Phenols; Phytoestrogens; Propionates; Pyrazoles; Rats; Rats, Long-Evans; Sexual Behavior, Animal

This study was designed to evaluate the effects of a novel phytoestrogen, α-zearalanol (α-ZAL), and estradiol benzoate (B-E₂), on c-myc, c-fos, and EGFR expression in normal human breast tissues implanted into nude mice. A xenograft-model, pieces of normal human breast tissue implanted subcutaneously into 9-10-week-old athymic nude mice, was established. The mice were divided into five groups subjected to the following treatments: normal saline (Controls); α-ZAL at 1 and 5 mg/kg; and estradiol benzoate (B-E₂) at 1 and 5 mg/kg. Treatment was given every other day, and human breast tissues were removed for experiments after treatment for 30 days. The expression of c-myc, c-fos, and EGFR mRNAs were determined by in situ hybridization. α-ZAL decreased expression of c-myc (p < 0.05). About 1 mg/kg α-ZAL increased EGFR expression (p < 0.05) and two dosage of α-ZAL increased c-fos expression (p < 0.01) compared with control. B-E₂ significantly increased expression of c-myc, c-fos, and EGFR mRNAs expression compared with controls (p < 0.01). The extents of the increases in EGFRmRNA expression induced by α-ZAL and in c-fos mRNA by 5 mg/kg α-ZAL were lower than those induced by B-E₂ (p < 0.01). These data suggest that the phytoestrogen α-ZAL may be safer than estrogen on breast.

Topics: Animals; Breast; ErbB Receptors; Estradiol; Female; Humans; In Situ Hybridization; Mice; Mice, Inbred BALB C; Mice, Nude; Phytoestrogens; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myb; RNA, Messenger; Zeranol

This study was designed to assess the effect of the phytoestrogenic compound ferutinin, chronically administered in ovariectomized progesterone primed rats, alone or in combination with estradiol benzoate. After 2, 3 and 4 weeks of treatments, female rats were tested for receptive (lordosis) and proceptive behaviors (hops, darts and ear wigglings). Ferutinin given alone markedly increased the intensity of the lordotic response in ovariectomized rats but failed to significantly affect proceptivity. On the other hand estradiol benzoate significantly increased both receptive and proceptive behaviors. When administered in combination with estradiol, ferutinin reduced the increase in receptivity and proceptivity due to estrogen effects, acting as an antiestrogen. At the end of the behavioral experiments, animals were sacrificed and Western blot analysis of estrogen receptor alpha (ERalpha) levels was performed in the dissected hypothalami. Ferutinin increased ERalpha expression when administered alone, as estradiol did, but decreased the response to estradiol when administered in combination. These results suggest that ferutinin displays estrogenic or antiestrogenic activity through ERalpha in the hypothalamus, depending on the absence or the presence of estrogen priming.

Topics: Animals; Benzoates; Bridged Bicyclo Compounds; Cycloheptanes; Estradiol; Estrogen Receptor alpha; Female; Hypothalamus; Male; Ovariectomy; Phytoestrogens; Rats; Rats, Sprague-Dawley; Sesquiterpenes; Sexual Behavior, Animal

The modulatory influence of the phytoestrogen biochanin A, an isoflavinoid found in red clover (Trifolium pratense), on the differentiation and proliferation of mammary epithelial cells and the expression of estrogen receptor-alpha (ER-alpha) in female prepubertal Sprague-Dawley rat mammary glands was examined, for which there have been no reports to date. Biochanin A (500 microg/g bw) was injected subcutaneously on days 16, 18 and 20 post-partum. The mammary gland was dissected out and terminal end buds, terminal ducts and lobules were counted. ER-alpha, Bcl2, Bax and caspase-3 expression were determined by immunohistochemistry. Estradiol benzoate (EB) (500 ng/g bw) and dimethyl sulphoxide (DMSO) were used as the reference and vehicle, respectively. The results showed a significant enhancement of differentiation at post-natal day (PND) 21 as well as at PND 50 in the mammary glands. There was a significant decrease of ER-alpha expression at PND 21 and an increased expression of the same at PND 50, whereas increased proliferation at PND 21 and increased apoptosis at PND 50 in the mammary glands were observed in biochanin A treated animals. The mode and magnitude of the effect of biochanin A was almost similar to that of EB. These findings suggested that prepubertal exposure to biochanin A modulated the regulatory processes and in turn enhanced the differentiation and development of mammary glands in female rats. These observations may have significance in human health.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Differentiation; Estradiol; Estrogen Receptor alpha; Female; Genistein; Mammary Glands, Animal; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley

Neonatal exposure to endocrine disrupting compounds (EDCs) can impair reproductive physiology, but the specific mechanisms by which this occurs remain largely unknown. Growing evidence suggests that kisspeptin (KISS) neurons play a significant role in the regulation of pubertal onset and ovulation, therefore disruption of KISS signaling could be a mechanism by which EDCs impair reproductive maturation and function. We have previously demonstrated that neonatal exposure to phytoestrogens decreases KISS fiber density in the anterior hypothalamus of female rats, an effect which was associated with early persistent estrus and the impaired activation gonadotropin releasing hormone (GnRH) neurons. The goals of the present study were to (1) determine if an ERalpha selective agonist (PPT) or bisphenol-A (BPA) could produce similar effects on hypothalamic KISS content in female rats and (2) to determine if male KISS fiber density was also vulnerable to disruption by EDCs. We first examined the effects of neonatal exposure to PPT, a low (50 microg/kg bw) BPA dose, and a high (50 mg/kg bw) BPA dose on KISS immunoreactivity (-ir) in the anterior ventral periventricular (AVPV) and arcuate (ARC) nuclei of adult female rats, using estradiol benzoate (EB) and a sesame oil vehicle as controls. AVPV KISS-ir, following ovariectomy (OVX) and hormone priming, was significantly lower in the EB and PPT groups but not the BPA groups. ARC KISS-ir levels were significantly diminished in the EB and high dose BPA groups, and there was a nonsignificant trend for lower KISS-ir in the PPT group. We next examined effects of neonatal exposure to a low (50 microg/kg bw) dose of BPA and the phytoestrogens genistein (GEN) and equol (EQ) on KISS-ir in the AVPV and ARC of adult male rats, using OVX females as an additional control group. None of the compounds affected KISS-ir in the male hypothalamus. Our results suggest that the organization of hypothalamic KISS fibers may be vulnerable to disruption by EDC exposure and that females might be more sensitive than males.

Topics: Animals; Animals, Newborn; Benzhydryl Compounds; Estradiol; Estrogen Receptor alpha; Female; Hypothalamus; Kisspeptins; Male; Nerve Fibers; Ovariectomy; Phenols; Phytoestrogens; Progesterone; Proteins; Pyrazoles; Rats; Rats, Long-Evans; Sex Characteristics

Oestrogen deficiency leads to a considerable bone loss, thus, osteopenia and osteoporosis are serious complications after menopause.. 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.. 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.. 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.. In spite of its mild uterotropic potential, dietary equol shows limited bone sparing effects in ovx rats.

Topics: Animals; Bone Density; Dietary Supplements; Equol; Estradiol; Female; Isoflavones; Osteoporosis; Ovariectomy; Phytoestrogens; Phytotherapy; Rats; Rats, Sprague-Dawley

High doses of estradiol (E(2)) can impair spatial learning in the Morris water maze, in ovariectomized mice, but the same dose has no effect on adult castrated males. Here, we test the hypothesis that this sex difference is caused by neonatal actions of E(2). In Experiment 1, C57BL/6J pups were given daily estradiol benzoate (EB) or oil injections from the day of birth until postnatal Day 3. Adults were gonadectomized and received EB (s.c.) or oil 28 h before the first day of training, and 4 h before each of four daily training sessions on the Morris water maze. Females given oil as neonates, and EB prior to training displayed the poorest performance. Females that received EB as neonates and EB prior to training were insensitive to the deleterious effects of adult EB and performed better than males given the same hormone treatments. We conducted a second experiment using aromatase enzyme knockout (ArKO) mice. Adult male and female ArKO and wild-type (WT) littermates were gonadectomized and received either injections of oil or EB prior to and during water maze training (as described above). Hormone treatment failed to affect performance, yet, female but not male ArKO mice showed impaired learning compared to WT littermates. Thus, exposure to estradiol during neonatal development can counteract the deleterious effects of EB on adult spatial learning.

Topics: Animals; Animals, Newborn; Aromatase; Diet; Estradiol; Female; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Phytoestrogens; Spatial Behavior

The pharmacokinetics and time course actions of the soy isoflavone, genistein, and estradiol benzoate (EB) on sex steroid and growth factor signaling were compared in the rat uterus. Following one s.c. injection of 500 mg genistein/kg BW or 500 microg EB/kg BW, AUC for genistein was 20171.8 ng h/ml and was 15.7 ng h/ml for estradiol-17-beta. Estrogen receptor-alpha (ER-alpha) decreased within 2 h of genistein or EB treatment, returning to basal levels within 24 and 48 h, respectively. In response to genistein and EB, progesterone receptors (PRs) A and B increased between 16 and 24 h, with a significant increase at 24 and 48 h. Epidermal growth factor receptor (EGFR) expression peaked 16 h after genistein or EB treatment, inversely correlating with extracellular regulating kinase (ERK) phosphorylation. These effects were inhibited by antiestrogen pretreatment, demonstrating a requirement for ER. At 16 h, uterine weight, epithelial cell height, and cell proliferation increased. While EGFR levels increased, phosphorylated EGFR was not altered. Reduced phosphorylation of downstream kinases corresponded with decreased stromal phosphorylated-ERK (P-ERK) immunolabeling, suggesting signal attenuation. Dynamic profiling of sex steroid receptors and EGF signaling molecules suggest a similar mechanism of action for genistein and EB in the uterus, albeit at approximately 1000-fold concentration.

Topics: Animals; Anticarcinogenic Agents; Area Under Curve; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Gene Expression Regulation; Genistein; Immunoblotting; Immunohistochemistry; Injections, Subcutaneous; Organ Size; Phosphorylation; Phytoestrogens; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Signal Transduction; Uterus

We evaluated the mechanism of action by the phytoestrogen genistein in the prepubertal rat uterus, when administered pharmacologically or physiologically. Female rats were injected with genistein (500 microg/g body weight), estradiol benzoate (EB) (500 ng/g body weight) or vehicle, dimethylsulfoxide (DMSO), on days 16, 18, and 20 postnatal. In 21-day-old rats, both compounds increased circulating estradiol and decreased progesterone concentrations. Uterine estrogen receptor alpha (ER-alpha) and androgen receptor (AR) proteins were reduced, and progesterone receptors (PR) were increased, as measured by western blot analyses. Immunohistochemistry for ER-alpha was confirmatory. Reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated a decrease in ER-alpha, but not in ER-beta, PR and AR mRNA levels following genistein treatment. In prepubertal rats exposed perinatally to 250 mg genistein per kg AIN-76A diet or 250 microg estradiol per kg diet, uterine ER-alpha, AR, and PR proteins were not altered significantly. We conclude that pharmacologic, but not physiologic concentrations of genistein can modulate sex steroid receptor expression in the rat uterus.

Topics: Animals; Body Weight; Estradiol; Estrogens, Non-Steroidal; Female; Gene Expression Regulation; Genistein; Immunohistochemistry; Isoflavones; Organ Size; Ovariectomy; Ovary; Phytoestrogens; Plant Preparations; Progesterone; Rats; Rats, Sprague-Dawley; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sexual Maturation; Testosterone; Uterus