Weak Estrogenic Activity Research Articles

Screening the foods of an endangered parrot, the kakapo (Strigops habroptilus), for oestrogenic activity using a recombinant yeast bioassay.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

Endocrine effects of the podophyllotoxine derivative drug CPH 82 (Reumacon®) in patients with rheumatoid arthritis

CPH 82 is a non-steroid antirheumatic drug containing two benzylidenated podophyllotoxin glucosides with no affinity for the glucocorticoid receptor. Treatment with CPH 82 as single drug therapy significantly decreased serum and urinary cortisol and cortisol metabolites, serum adrenal androgens and urinary androgen metabolites, plasma ACTH and serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), and increased serum levels of sex hormone-binding globulin (SHBG). Significant positive correlations were found between serum TNF-alpha and plasma ACTH and between serum IL-6 and TNF-alpha on the one hand and serum and urinary cortisol and cortisol metabolites on the other. The initial action of CPH 82 on adrenal steroidogenesis may be a reduction in cytokine levels due to the drugs' antiinflammatory effect. This causes decreased ACTH stimulation, resulting in a reduced adrenocortical steroid secretion. Accumulation of the drug in the adrenal cortex may also affect adrenal steroidogenesis. The elevated SHBG levels may be caused by a weak estrogenic activity of the drug.

Prepubertal exposure to compounds that increase prolactin secretion in the male rat: effects on the adult prostate.

To test the hypothesis that a transient increase in prolactin (PRL) secretion prior to puberty can result in an alteration of the adult prostate, male rats were exposed from postnatal Days (PND) 22 to 32 to compounds that increase PRL secretion. These compounds included pimozide (a dopamine antagonist), estradiol-17beta, and bisphenol A (a monomer of polycarbonate plastics reported to have weak estrogenic activity). During dosing, pimozide (PIM), bisphenol A (BPA), and estradiol-17beta (E(2)) stimulated an increased secretion of PRL. At 120 days of age, the lateral prostate weight was increased in the PIM and BPA groups as compared to the vehicle-injected controls. Examination of the prostates revealed inflammation in the lateral lobes of all treated groups. Results of a myeloperoxidase assay, a quantitative assay to assess acute inflammation, indicated an increase in the percentage of males with neutrophil infiltrate in the lateral prostates of the PIM and E(2) treatment groups compared to their respective controls. The histological evaluations of these tissues confirmed an increase in luminal polymorphonuclear cells and interstitial mononuclear cells of the lateral prostates in all treatment groups. Administration of the dopamine agonist, bromocriptine, to the estradiol-implanted males from PND 22 to 32 reversed the induction of lateral prostate inflammation by estradiol, suggesting that PRL was necessary for the inflammatory effect. This study demonstrates that prepubertal exposures to compounds that increase PRL secretion, albeit through different mechanisms, can increase the incidence of lateral prostate inflammation in the adult.

Estrogenic activity of glycitein, a soy isoflavone.

Glycitein (4',7-dihydroxy-6-methoxyisoflavone) accounts for 5-10% of the total isoflavones in soy food products. The biological activity of this compound has not been reported to date, although numerous studies have been performed with the other soy isoflavones, daidzein and genistein. Glycitein was isolated from soy germ to 99% purity. Weaning female B6D2F1 mice were dosed with glycitein (3 mg/day), genistein (3 mg/day), and diethylstilbestrol (DES) (0.03 microg/day) in 5% Tween 80 by gavage for 4 days. A control group received an equal volume of 5% Tween 80 solution daily. The uterine weight increased 150% with glycitein (p < 0.001), 50% with genistein (p < 0. 001), and 60% with DES (p < 0.001) compared with the control group. DES, 17beta-estradiol, and three isoflavones (daidzein, genistein, and glycitein) were examined for their competitive binding abilities with 17beta-((3)H)estradiol to the estrogen receptor proteins of the B6D2F1 mouse uterine cytosol. The concentrations of each compound required to displace 50% of the ((3)H)estradiol at 5 nM in the competitive binding assay were 1.15 nM DES, 1.09 nM 17beta-estradiol, 0.22 microM genistein, 4.00 microM daidzein, and 3.94 microM glycitein. These data indicated that glycitein has weak estrogenic activity, comparable to that of the other soy isoflavones but much lower than that of DES and 17beta-estradiol.

In VitroEstrogenicity of the Catechol Metabolites of Selected Polychlorinated Biphenyls

A considerable body of work has demonstrated that phenolic polychlorinated biphenyl (PCB) metabolites, structural analogues to estradiol, bind to the soluble estrogen receptor (ER) and that hydroxy PCB-ER complexes will translocate into the nucleus and bind to ER response elements in cultured cells. Although catechol estrogens exhibit weak estrogenic activity, the catechol PCB metabolites which are structurally similar to these ER agonists have gone untested for potential estrogenicity. In the present work we have assessed the estrogenicity of this second group of PCB metabolites, the catechols. The test compounds used in the present study were chosen to elucidate the effects of chlorine and catechol position onin vitroestrogenicity. Cultured HeLa cells, transfected with the estrogen reporter gene ERET81CAT and mouse ER cDNA, were incubated with PCB catechols. The cells were harvested at 28 h posttransfection and assayed for chloramphenicol acetyl transferase (CAT) activity. The responses elicited by the PCB catechols tested fell within the range of effect measured for the catechol estrogens and phenolic PCBs, and were within the range previously reported for other “environmental estrogens” such as nonylphenol and o,p′-DDT. Maximal measured responses were achieved at concentrations approximately two to three orders of magnitude higher than that of 17-β-estradiol, indicating that PCB catechols have estrogenic activityin vitro.The extent of chlorination and the position of the catechol (3,4 vs 2,3 substitution) were important in determining estrogenicity in the compounds tested. The 2,3-catechol showed no detectable activity in this system, while activity of the 3,4-catechols increased with the degree of chlorination. The observed estrogenicity of PCB catechols suggests that further oxidative metabolism of estrogenic PCB phenolic metabolites would not necessarily result in lowering the total estrogenic burden of a PCB-exposed organism. The present results imply that if estrogenic activity is assigned to an individual phenol, the potential contribution of its catechol metabolites to the total estrogenic burden should also be taken into consideration.

Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals

The E-Screen assay serves as an in vitro tool for the detection of estrogenic activity of chemicals and extracts of environmental samples. Based on the induction of proliferation in human estrogen receptor-positive MCF-7 breast cancer cells we could substantially simplify the assay. As one important step of validation we applied the modified assay for testing nine known xenoestrogens. We could confirm the results of other groups assuring the reproducibility of the E-Screen assay. The results provide evidence that the E-Screen assay is suitable for determination of estradiol equivalency factors (EEFs) for environmental estrogens to rank their estrogenic potency relative to the natural estrogen 17β-estradiol. Further, we used the optimized proliferation test to screen nine halogenated phenolic compounds for their possible estrogenic potency. Three widely applied chemicals expressed a weak receptor-mediated estrogenic activity: the flame retardant Tetrabromo-Bisphenol-A, the disinfectant 4-chloro-3-methylphenol, and the herbicide educt 4-chloro-2-methylphenol. Their estrogenic potencies were five to six orders of magnitude lower than that of 17β-estradiol.

Phytoestrogens: The Food of the Menopause?

Objectives: to provide an overview of the literature on the subject of phytoestrogens, with particular emphasis on their role in menopause, and to a lesser extent, on women’s health.Data Sources: a ten-year Medline search using phytoestrogens as both a subject and a textword. The author’s personal files and the reference lists from selected articles were explored. The Internet was selectively explored.Study Selection: review articles and those dealing with women’s health or menopausal issues were selected as were other studies if they shed light on the primary objective.Data Extraction: data were heterogeneous and not appropriate for formal analysis.Results: the phytoestrogens are non-steroidal, diphenolic compounds that occur naturally in plants. Soybeans and flaxseed both contain large amounts of isoflavones and lignans, which are metabolized in the gut to compounds which bind to the estrogen receptor and have weak estrogenic or anti-estrogenic activity. Epidemiological evidence of a lower incidence of menopausal symptoms, breast, endometrial and prostate cancer, and of a lower rate of cardiovascular disease in populations consuming large amounts of the phytoestrogens in their diet, has led to interest in their preventive benefits. Phytoestrogens appear to increase sex hormone binding globulin levels, compete for aromatase and inhibit tyrosine kinase. Individuals vary greatly in how they metabolize and absorb these substances. Clinically, such substances may reduce the severity of menopausal flushing.Conclusions: physicians seeing menopausal women should be aware of these compounds and of the type and amount of dietary supplements their patients may be taking.

Isoflavonoids and Lignans in Legumes: Nutritional and Health Aspects in Humans

Dietary factors are considered important environmental risk determinants for Western diseases. Studies have revealed beneficial or protective effects of the consumption of legumes with regard to hypercholesterolaemia and coronary heart disease, obesity, diabetes mellitus, and menopause. During the last decade attention has been focused on soy and soybean products. Several constituents have been isolated: isoflavones, phytosterols, protease inhibitors, inositol hexaphosphate, and saponins. Our interest concentrates on hormone-like bisphenolic phytoestrogens of dietary origin, the lignans and isoflavonoids. Their glycosides, converted by gut bacteria to mammalian derivatives with weak estrogenic and antioxidative activity, originate in leguminous seeds. We developed an isotope dilution gas chromatography-mass spectrometry method for quantitative determination of the isoflavones, formononetin, biochanin A, daidzein, genistein and coumestrol, and the lignans secoisolariciresinol and matairesinol, in food samples. We measured the four isoflavonoids and coumestrol, and, for the first time, the two lignans in 52 leguminous seeds and found high concentrations of isoflavonoids (0–1853.35 mg/kg; 0–7.3 mmol/kg dw) but lower amount of lignans (0–15.85 mg/kg; 0.05 mmol/kg dw). The highest plasma levels of their metabolites are found in individuals living in countries or regions with low cancer and cardiovascular disease incidence and these are probably sufficient to influence intracellular enzymes, protein synthesis, growth factor action, malignant cell proliferation, differentiation, and angiogenesis. Leguminous seeds, therefore, in respect to their abundant concentrations of phytoestrogens, are strong candidates for a role as natural cancer-protective food.

Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol.

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although [3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the "Estriol Hypothesis" on the protective role for estriol in opposing carcinogenic effects of estradiol.

Molecular and Kinetic Basis for the Mixed Agonist/Antagonist Activity of Estriol

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although[ 3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the “Estriol Hypothesis” on the protective role for estriol in opposing carcinogenic effects of estradiol.

The estrogenic activity of phthalate esters in vitro.

A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen. a selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells. A small number of the commercially available phthalates tested showed extremely weak estrogenic activity. The relative potencies of these descended in the order butyl benzyl phthalate (BBP) > dibutyl phthalate (DBP) > diisobutyl phthalate (DIBP) > diethyl phthalate (DEP) > diisiononyl phthalate (DINP). Potencies ranged from approximately 1 x 10(6) to 5 x 10(7) times less than 17beta-estradiol. The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells. Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays. A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mon-n-octyl phthalate; all were wound to be inactive. One of the phthalates, ditridecyl phthalate (DTDP), produced inconsistent results; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive. analysis by gel chromatography-mass spectometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho-isomer of bisphenol A. It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o,p'-bisphenol A--in the yeast screen; hence, DTDP is probably not weakly estrogenic. The activities of simple mixtures of BBP, DBP, and 17beta-estradiol were assessed in the yeast screen. No synergism was observed, although the activities of the mixtures were approximately additive. In summary, a small number of phthalates are weakly estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vivo; this will require tests using different classes of vertebrates and different routes of exposure.

In vitro synergistic interaction of alligator and human estrogen receptors with combinations of environmental chemicals.

The effect of mixtures of environmental chemicals with hormonal activity has not been well studied. To investigate this phenomenon, the estrogen receptor (ER) from the American alligator (aER) or human (hER) was incubated with [3H]17beta-estradiol in the presence of selected environmental chemicals individually or in combination. The environmental chemicals included the insecticide chlordane, which has no estrogenic activity, and the pesticides dieldrin and toxaphene, which have very weak estrogenic activity. Chlordane, dieldrin, and toxaphene individually demonstrated no appreciable displacement of [3H]17beta-estradiol from aER and hER at the concentration tested. A combination of these chemicals inhibited the binding of [3H]17beta-estradiol by 20 to 40%. Alachlor, a chemical recently discovered to have weak estrogenic activity, also displaced [3H]17beta-estradiol more effectively in combination with dieldrin than alone. These results indicate that combinations of some environmental chemicals inhibit [3H]17beta-estradiol binding in a synergistic manner. This suggests that the ER may contain more than one site for binding environmental chemicals. The possibility that the ER binds multiple environmental chemicals adds another level of complexity to the interaction between the environment and the endocrine system.

Weak estrogenic activity from continuous-release pellets

In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17β-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.

Effect of tamoxifen on lipids and lipid metabolising marker enzymes in experimental atherosclerosis in Wistar rats.

Tamoxifen, a non-steroidal anti-oestrogen, is used in the treatment of breast cancer, both receptor positive and negative tumours. It also possesses weak oestrogenic activity which forms the basis of this study. Tamoxifen (2 different dosages) was administered through diet (10 mg/kg diet and 20 mg/kg diet) to experimental atherosclerosis induced female rats to assess the effect of tamoxifen on plasma lipid levels, lipoprotein cholesterol level and on the activity of lipid metabolising enzymes. The plasma total lipid level was increased in atherosclerosis suffering animals compared to control animals with concomitant changes in the activity of lipid metabolising enzymes. HDL-cholesterol was decreased while LDL-cholesterol and VLDL-cholesterol were increased in the atherosclerosis induced group. Cholesterol and free cholesterol were decreased in tamoxifen treated groups while the other lipids show a moderate increase. HDL-cholesterol was increased but LDL-cholesterol was decreased in the tamoxifen treated groups. The higher dosage tamoxifen given group animals show significantly favourable results from therapy stand point when compared to diseased group.

Profound effects of the weak environmental estrogen-like chemical bisphenol A on the growth of the mammary gland of Noble rats.

In the present study we have examined the effects of the environmental estrogenic chemical bisphenol A on proliferative activity, cell cycle kinetics and differentiation of the mammary gland of female Noble rats. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased in response to exposure to both low (0.1 mg/kg/day) and high (54 mg/kg/day) doses of bisphenol A compared to controls. The proliferative activity of epithelial cells was increased by 143% over controls by the exposure of animals to the low dose of bisphenol A, whereas a 220% increase over controls was observed for the high dose of bisphenol A. The labelling index and growth fraction were 19% and 27%, respectively, for a low dose of bisphenol A; and 27% and 45%, respectively, for a high dose of bisphenol A, compared to 18% and 31%, respectively, in controls. A significant increase in the conversion of mammary epithelial cells from G0, to G1, and S-phase cells by 1.8 and 4.5-fold, respectively, was observed in animals exposed to the high dose of bisphenol A compared to that of controls. Based on the previously reported estrogenic activity of an equivalent dose of bisphenol A to that of diethylstilbestrol (DES) (0.1 mg/kg/day), a calculated theoretical dose of the order of 10(6)-fold higher of bisphenol A will be required to produce the same biological effects as DES. A comparison of the proliferative activity of DES and that of an equivalent dose of bisphenol A observed in this study, however, revealed that its influence on proliferative activity in the epithelial cells of the mammary gland was profound. The weak estrogenic activity of bisphenol A does not explain its profound effect on cell proliferation observed in this study. Perturbation of the cell cycle is considered a risk factor for the development of cancer. Bisphenol-mediated perturbation of the cell cycle in epithelial cells may produce adverse effects in the mammary glands of Noble rats.

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Identification of Environmental Chemicals with Estrogenic Activity Using a Combination of In Vitro Assays

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Bone targeted drugs 2. synthesis of estrogens with hydroxyapatite affinity

The utility of the bone targeting 4-carboxy-3-hydroxy-1,2-pyrazole heterocycle was tested by the synthesis of hybrids with the non-steroidal estrogen hexestrol. Compounds 1 and 2 demonstrated significant hydroxyapatite affinity, while maintaining weak estrogenic activity in whole cell assays.

Exposure of environmental estrogenic compound nonlyphenol to noble rats alters cell-cycle kinetics in the mammary gland

p-Nonylphenol is an environmental estrogen-like chemical. Nonylphenol has previously been shown to mimic the actions of estrogen both in vivo and in vitro. In the present study, we have examined the effects of an environmental estrogenic chemical, nonlylphenol, on the proliferative activity, cell-cycle kinetics, and differentiation of the mammary gland of female Noble rats. The exposure of animals to two different doses of nonlylphenol (0.01 and 7.1 mg/24 h) significantly increased proliferation of the mammary epithelial cells. Both labeling index and growth fraction were increased by nonlylphenol treatment. Labeling index and growth fraction were 24 and 38%, respectively, for a low dose of nonylphenol; and 32 and 67%, respectively, for a high dose of nonlyphenol, compared to 18 and 35%, respectively, of controls. In addition, nonlylphenol exposure altered cell-cycle kinetics. Both low and high doses of nonylphenol significantly increased the conversion of mammary epithelial cells from G0 to G1 and S-phase cells by 2.2-and 2.6-fold, and by 11- and 4-fold, respectively, compared to that of controls. Differentiation measured by the degree of lobular maturation revealed that the conversion of immature structures to mature structures was significantly increased in response to nonylphenol exposure compared to that of control. Based on the previously reported estrogenic activity of an equivalent dose of nonylphenol to that of DES (0.01 mg/d), a calculated theoretical dose of the order of 10(5)- to 10(6)-fold higher of nonlylphenol will be required to produce the same biological effects as DES. However, the data of this study showed that exposure of 0.01 mg/d of nonlylphenol produced profound effect on cell proliferation in the mammary gland. The weak estrogenic activity of nonlylphenol does not explain its profound effect on cell proliferation observed in this study. Perturbation of cell cycle is considered as a risk factor for the development of cancer. Changes in proliferation and cell cycle have been shown to lead to genetic instability, ultimately resulting in cell transformation. Our results indicated an increase in labeling index and growth fraction, and a perturbation in cell-cycle kinetics from nonlylphenol exposure. Perturbation of cell cycle in response to nonlylphenol exposure may produce adverse effects in the mammary glands of the Noble rats.

Naringenin: A weakly estrogenic bioflavonoid that exhibits antiestrogenic activity

Treatment of immature 21-day-old female Sprague-Dawley rats with 17β-estradiol (E2) (0.5 μg/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 μg/rat) plus naringenin (30 mg/rat), there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [ 32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1–1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 μM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.