Kevin L. Medlock

Estradiol Down-Regulation of the Rat Uterine Estrogen Receptor

Abstract We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighed, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radio-immunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.

The Effects of Phytoestrogens on Neonatal Rat Uterine Growth and Development

Abstract Phytoestrogens found in clover, alfalfa, and soybeans have caused reproductive toxicity in several mammalian species. Other estrogens, such as diethylstilbestrol (DES), are developmental toxicants, reducing uterine estrogen receptor (ER) concentration, altering uterine growth, and eliciting reproductive tract abnormalities in the rat. The present study examines the effects of the phytoestrogens coumestrol and equol on the developing rat uterus. Various doses of these compounds were injected sc on postnatal days (PND) 1-5 or 1-10 to ascertain their effects on uterine weight and ER levels, and on PND 10-14 to determine their effects on uterine weight and gland genesis. Coumestrol (PND 1-5) was about 10-3 as potent as DES in increasing uterine weight (wet or dry) while equol increased dry weight only, with a potency of 10-5 that of DES. Although the 10 and 100 μg doses of coumestrol (PND 1-5 or 1-10) initially increased uterine wet weight, by PND 20 uterine weights either equaled or fell significantly below controls. The 100-μg dose of coumestrol (PND 1-5 or 1-10) reduced ER levels at all ages, while the 10-μg dose was not as effective. Equol (PND 1-5 or 1-10) did not affect ER levels. Premature uterine gland genesis occurred by PND 9 for the PND 1-5 100-μg coumestrol dose. When given on PND 10-14 (the critical period of gland genesis), 10 μg and 100 μg of coumestrol and 10 μg DES greatly increased uterine weight, while no effect was elicited by equol. Although coumestrol and equol inhibited uterine gland genesis in a dose-dependent manner, neither abolished gland genesis as did 10 μg of DES or tamoxifen. These data demonstrate that coumestrol elicits uterine biochemical and morphological toxicity much like DES. Equol decreased uterine gland number without increasing uterine wet weight or luminal epithelial hypertrophy, which is inconsistent with either an estrogenic or antiestrogenic action in the uterus.

Methylation Profile and Amplification of Proto-Oncogenes in Rat Pancreas Induced with Phytoestrogens

Abstract Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 μg of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras protooncogene. No hypermethylation or hypomethylation was observed in the protooncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders.

Effects of postnatal des treatment on uterine growth, development, and estrogen receptor levels

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.

Flavonoids Inhibit Estrogen Binding to Rat Alpha-Fetoprotein

Abstract There is considerable interest in the role(s) of plant-derived compounds such as bioflavonoids in regulating steroid hormone action in mammals, and in particular, the possible effects of the bioflavonoids on the growth of steroid-dependent breast and prostate tumors and on possible abnormal development of steroid-sensitive tissues. Studies of the hormone-like actions of bioflavonoids often use fetal or neonatal rats, which contain high levels of serum alpha-fetoprotein (AFP), a protein that binds estradiol with a K d ~ 5 × 10−9 M. Interaction of bioflavonoids with AFP could affect the availability of estrogens to estrogen-responsive cells, as well as the actions of bioflavonoids. These considerations motivated us to study the effect of several flavonoids (quercetin, rutin, naringenin, chrysin, apigenin, kaempferol, myricetin, morin, fisetin) and isoflavonoids (daidzein, genistein) on estrogen binding to rat AFP. We found that naringenin, a flavanone, and quercetin and kaempferol, flavonols, inhibit estrogen binding to AFP with apparent K ds of about 5 × 10−7 M. To our surprise, the two isoflavonoids, daidzein and genistein, have K ds of about 5 × 10−6 M for AFP. This 1Q-fold difference in affinity for AFP between flavonoids and isoflavonoids suggests that AFP has a specificity for the flavonoid structure. Moreover, the affinities of bioflavonoids for rat AFP are sufficiently high to suggest that flavonoids and isoflavonoids could modulate estradiol and estrone binding to rat AFP in vivo, when present at dietary levels. Additionally, the potency of the plant estrogens may be altered by binding to AFP. The flavonoids that we tested have different hydroxyl and glucoside substituents on the A, B, and C rings, which allows us to define some of the spatial requirements for binding to AFP. We find that 5,7-hydroxyl groups in ring A and a 4′-hydroxyl group in ring B are important for binding to AFP. This information, combined with molecular modeling studies, may elucidate the molecular basis for recognition of flavonoids and estrogens by AFP. Also, these findings indicate that the flavonoid levels in the diet need to be considered in studies of the effects of various xenobiotics and endocrine manipulations on experimental animals, particularly during development when serum estrogen binding protein concentrations are often elevated. Finally, bioflavonoids should be useful tools for understanding the variety of estrogen actions initiated by different structural classes of estrogens.

PROGESTERONE AND ESTRADIOL INTERACTION IN THE REGULATION OF RAT UTERINE WEIGHT AND ESTROGEN RECEPTOR CONCENTRATION

Abstract Autologous down-regulation of hormone receptors has been shown for several steroid hormones. We have previously shown estradiol (E2) regulates estrogen receptor (ER) in ovariectomized (OVX) rats. These studies have been extended to investigate the interaction between progesterone (P) and E2 in the regulation of ER and uterine weight. We implanted Silastic capsules containing varying concentrations of E2 (0.0005 mg E2/ml to 5.0 mg E2/ml of sesame oil) into adult female Sprague-Dawley rats one week after OVX. Simultaneously with implantation, P injections were started (sc in sesame oil) at doses of 1-40 mg/day for three days. In the absence of P, the 0.05 mg E2/ml implant significantly increased total ER levels (measured by cytosol and nuclear exchange assays) by 25%, while the two highest concentrations of E2 (0.5 and 5.0 mg E2/ml) significantly decreased cytosol and total ER levels by at least 40%. No P dose altered ER levels in OVX rats or in rats given E2 implants of 0.01 mg E2/ml or lower. At E2 implant concentrations of 0.05 mg E2/ml and higher, P decreased total ER levels 30%-50% compared to the appropriate E2-only controls. P increased uterine weight by 25% in OVX controls and in rats treated with E2 implants of 0.01 mg E2/ml and below. In contrast, P inhibited uterine weight gain induced by 0.05-5.0 mg/E2 ml implants by 20%-30%; maximal inhibition occurred at 5 mg/day of P and above. These data demonstrate that P increases uterine weight but does not alter ER concentration in rats with low E2 levels (OVX or low E2 concentration implants) but decreases uterine weight and down-regulates ER at higher E2 levels, regardless of whether ER is upregulated or down-regulated by E2. [P.S.E.B.M. 1994, Vol 205]

The postnatal ontogeny of the rat uterine estrogen receptor

Abstract Rat uterine cytosol and nuclear estrogen receptor (ER) levels and uterine weight were determined on postnatal days 3, 5, 7, 10, 1Z 15, 18, 21 and 28. Rats were injected with sesame oil (controls) or with 1 mg/kg diethylstilbestrol (DES) and uteri removed 1 h later for ER determination. DES injection translocated cytosol receptor to the nucleus, allowing measurement by nuclear exchange assay (NEA) in the absence of alphafetoprotein, an estrogen binding contaminant of neonatal uterine cytosols. Control animals generally gave curvilinear Scatchard plots which could be resolved into high affinity ( K d = 1.24 ± 0.16nM) estrogen receptor sites and low affinity ( K d = 10–40nM) sites. Nuclear ER in control animals increased rapidly from day 3 to day 12 (to 0.030 ± 0.003 pmol/100 μg DNA) and increased slightly thereafter. Cytosol ER increased from day 3 to a maximum at day 10 (0.490 ± 0.026 pmol/100 μg DNA) and decreased thereafter. DES-injected animals exhibited linear Scatchard plots, indicating one major binding component ( K d = 3.21 ± 0.15 nM). Relative uterine weight on days 1–9 in untreated animals (5.18 ± 0.11 mg/10 g b.wt.) increased to 7.04 ± 0.37 mg/10 g b.wt. on days 11–14. Increased uterine weight and cytosol and nuclear receptor levels around days 10–14 suggest that the uterus is responsive to endogenous estrogens during this period.

Chronic dose-response studies in mice fed 2-AAF.

Large numbers of mice were exposed to 2‐acetylaminofluorene (2‐AAF) in the diet in order to provide quantitative information on carcinogenic dose‐response relationships. Weanling BALB/c and C57BL/6 mice of both sexes were fed 2‐AAF ad libitum in their diet for 18 months at concentrations of 0, 100, 250, and 500 ppm. Occurrence of a bladder tumor was considered as the endpoint for determination of dose‐response curves. Data were accumulated from mice that died during the 18‐month period, from moribund mice that were necropsied, and from mice necropsied at the terminal sacrifice after 18 months of exposure to the carcinogen. A dose response was noted for the body weights of all animals and for the mortality rate and bladder tumor development of the females. In the males the dose of 500 ppm of 2‐A A F appeared to be above the maximum tolerated dose and resulted in early death of the animals, a decreased tumor incidence, and the lack of a dose‐related response. Bladder tumor development in the females followe...

Identification of uterine nuclear type II estrogen binding sites in estrogen treated rats

Uterine nuclear fractions from estrogen-treated rats contain both the estrogen receptor and a lower affinity estrogen binding site (type II site). In Scatchard plots of estrogen binding, two types of curves are seen. The hook-shaped form is composed of a linear component (the estrogen receptor) and a convex component (the type II site) while the curvilinear form is resolvable into two linear binding species (the estrogen receptor and a secondary site). To clarify the relationship between the two forms, we examined the curvilinear form from immature rats injected for 4 days with estradiol (E2) for type II site properties. Like the hook-shaped type II, this form could be detected in a nuclear exchange assay at both 37 and 4 degrees C, but at neither temperature in the presence of reducing agent. Additionally, the steroid specificity of the curvilinear form was identical to the hook-shaped form. The hook-shaped form was found in both immature and ovariectomized adult rats implanted for 6 days with an E2-releasing Silastic capsule to provide pharmacological E2 levels. When uteri from implanted animals displaying the hook-shaped form were mixed in various ratios with uteri lacking type II sites, the curvilinear form was produced. Animals given an E2 implant for 3 days, followed by a 3 day hormone-free period showed a curvilinear form. In vivo E2 dose-response experiments showed the curvilinear form at low E2 doses and the hook-shaped form at the high dose and in implanted animals. We conclude that curvilinear Scatchard plots result from the presence of authentic type II at lower concentrations than those giving rise to the hook-shaped form.