Soy Phytoestrogen Genistein Research Articles

Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models.

Sertoli cells are essential for germ cell development and function. Their disruption by endocrine disrupting chemicals (EDCs) or drugs could jeopardize spermatogenesis, contributing to male infertility. Perinatal exposure to EDCs and acetaminophen (APAP) disrupts male reproductive functions in animals and humans. Infants can be exposed simultaneously to the dietary soy phytoestrogen genistein (GEN) and APAP used for fever or pain relief. Our goal was to determine the effects of 10-100 µM APAP and GEN, alone or mixed, on immature Sertoli cells using mouse TM4 Sertoli cell line and postnatal-day 8 rat Sertoli cells, by measuring cell viability, proliferation, prostaglandins, genes and protein expression, and functional pathways. A value of 50 µM APAP decreased the viability, while 100 µM APAP and GEN decreased the proliferation. Sertoli cell and eicosanoid pathway genes were affected by GEN and mixtures, with downregulation of Sox9, Cox1, Cox2, and genes relevant for Sertoli cell function, while genes involved in inflammation were increased. RNA-seq analysis identified p53 and TNF signaling pathways as common targets of GEN and GEN mixture in both cell types. These results suggest that APAP and GEN dysregulate immature Sertoli cell function and may aid in elucidating novel EDC and drug targets contributing to the etiology of male infertility.

Differentiation Patterns of Uterine Carcinomas and Precursor Lesions Induced by Neonatal Estrogen Exposure in Mice.

Developmental exposure to estrogenic chemicals is an established risk factor for cancer of the female reproductive tract. This increase in risk has been associated with disruption of normal patterns of cellular differentiation during critical stages of morphogenesis. The goal of this study was to document uterine epithelial phenotypes over time following neonatal treatment with the synthetic estrogen diethylstilbestrol (DES) or the soy phytoestrogen genistein (GEN) in female CD-1 mice. Both DES and GEN induced three distinct populations of abnormal endometrial epithelial cells: luminal (SIX1+/P63-/CK14-/CK18+), basal (SIX1+/P63+/CK14+/CK18-), and mixed/bipotential (SIX1+/P63-/CK14+/CK18+), which were all established by early adulthood. In older animals, DES and GEN resulted in uterine carcinomas with mixed glandular, basal, and squamous cell elements. All carcinomas were composed largely of the three abnormal cell types. These findings identify novel epithelial differentiation patterns in the uterus and support the idea that disruption of cellular programming in early development can influence cancer risk later in life.

Endocrine Disruption of Vasopressin Systems and Related Behaviors.

Endocrine disrupting chemicals (EDCs) are chemicals that interfere with the organizational or activational effects of hormones. Although the vast majority of the EDC literature focuses on steroid hormone signaling related impacts, growing evidence from a myriad of species reveals that the nonapeptide hormones vasopressin (AVP) and oxytocin (OT) may also be EDC targets. EDCs shown to alter pathways and behaviors coordinated by AVP and/or OT include the plastics component bisphenol A (BPA), the soy phytoestrogen genistein (GEN), and various flame retardants. Many effects are sex specific and likely involve action at nuclear estrogen receptors. Effects include the elimination or reversal of well-characterized sexually dimorphic aspects of the AVP system, including innervation of the lateral septum and other brain regions critical for social and other non-reproductive behaviors. Disruption of magnocellular AVP function has also been reported in rats, suggesting possible effects on hemodynamics and cardiovascular function.

SIX1 Oncoprotein as a Biomarker in a Model of Hormonal Carcinogenesis and in Human Endometrial Cancer.

The oncofetal protein sine oculis-related homeobox 1 (SIX1) is a developmental transcription factor associated with carcinogenesis in several human cancer types but has not been investigated in human endometrial cancer. In a model of hormonal carcinogenesis, mice neonatally exposed to the soy phytoestrogen genistein (GEN) or the synthetic estrogen diethylstilbestrol (DES) develop endometrial cancer as adults. Previously, we demonstrated that SIX1 becomes aberrantly expressed in the uteri of these mice. Here, we used this mouse model to investigate the role of SIX1 expression in endometrial carcinoma development and used human tissue microarrays to explore the utility of SIX1 as a biomarker in human endometrial cancer. In mice neonatally exposed to GEN or DES, the Six1 transcript level increased dramatically over time in uteri at 6, 12, and 18 months of age and was associated with development of endometrial carcinoma. SIX1 protein localized within abnormal basal cells and all atypical hyperplastic and neoplastic lesions. These findings indicate that developmental estrogenic chemical exposure induces persistent endometrial SIX1 expression that is strongly associated with abnormal cell differentiation and cancer development. In human endometrial tissue specimens, SIX1 was not present in normal endometrium but was expressed in a subset of endometrial cancers in patients who were also more likely to have late-stage disease. These findings identify SIX1 as a disease biomarker in a model of hormonal carcinogenesis and suggest that SIX1 plays a role in endometrial cancer development in both mice and women. The SIX1 oncoprotein is aberrantly expressed in the endometrium following developmental exposure to estrogenic chemicals, correlates with uterine cancer, and is a biomarker in human endometrial cancers. Mol Cancer Res; 14(9); 849-58. ©2016 AACR.

The Effects of Phytoestrogen Genistein on Steroidogenesis and Estrogen Receptor Expression in Porcine Granulosa Cells of Large Follicles.

Genistein is a biologically active isoflavone with estrogenic or antiestrogenic activity which can be found in a variety of soy products. Since in pigs' diet soy is the main source of protein, genistein may affect the reproductive/endocrine systems in these animals. Genistein has been shown to alter porcine ovarian and adrenal steroidogenesis but the mechanism of this action is still not clear. It is known that genistein binds to both estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), although it has a higher affinity to ERβ. Moreover, this phytoestrogen was demonstrated to posses the activity of protein tyrosine kinase (PTK) inhibitor. The aim of the study was to examine the in vitro effects of genistein on: (1) progesterone (P4) and estradiol (E2) secretion by porcine luteinized granulosa cells harvested from large follicles, and (2) the mRNA and protein expression of ERa and ERβ in these cells. In addition, to verify the role of PTK-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. Genistein significantly inhibited P4 and did not affect E2 secretion by porcine luteinized granulosa cells isolated from large follicles. Lavendustin C did not affect basal steroids secretion by examined cells. Genistein did not alter ERa but increased ERβ mRNA levels in the cultured porcine granulosa cells. In contrast to medium follicles, the expression of ERβ protein was unaffected by genistein in granulosa cells of large follicles. To conclude, the soy phytoestrogen genistein acts directly on the porcine ovary to decrease progesterone production and to increase the expression of ERβ mRNA. Moreover, genistein-induced changes in follicular steroidogenesis and granulosal sensitivity to estrogens in pigs may depend on maturity of the follicles.

Soy phytoestrogen genistein increases liver deiodinase type 1 enzyme activity in the rat models of menopause and andropause

Soy phytoestrogen genistein increases liver deiodinase type 1 enzyme activity in the rat models of menopause and andropause

A diet containing the soy phytoestrogen genistein causes infertility in female rats partially deficient in UDP glucuronyltransferase

Soy beans contain genistein, a natural compound that has estrogenic effects because it binds the estrogen receptor with relatively high affinity. Genistein is therefore the most important environmental estrogen in the human diet.Detoxification of genistein is mediated through conjugation by UDP-glucuronyltransferase 1 and 2 (UGT1 and UGT2) isoenzymes.Gunn rats have a genetic deficiency in UGT1 activity, UGT2 activities are not affected. Because our Gunn rats stopped breeding after the animal chow was changed to a type with much higher soy content, we examined the mechanism behind this soy diet induced infertility. Gunn and control rats were fed diets with and without genistein. In these rats, plasma levels of genistein and metabolites, fertility and reproductive parameters were determined. Enzyme assays showed reduced genistein UGT activity in Gunn rats, as compared to wild type rats. Female Gunn rats were completely infertile on a genistein diet, wild type rats were fertile. Genistein diet caused a persistent estrus, lowered serum progesterone and inhibited development of corpora lutea in Gunn rats.Concentrations of total genistein in Gunn and control rat plasma were identical and within the range observed in humans after soy consumption. However, Gunn rat plasma contained 25% unconjugated genistein, compared to 3.6% in control rats. This study shows that, under conditions of reduced glucuronidation, dietary genistein exhibits a strongly increased estrogenic effect. Because polymorphisms that reduce UGT1 expression are prevalent in the human population, these results suggest a cautionary attitude towards the consumption of large amounts of soy or soy supplements.

Testing Chemicals for Effects on Breast Development, Lactation, and Cancer

Testing Chemicals for Effects on Breast Development, Lactation, and Cancer

Paracrine-Acting Adiponectin Promotes Mammary Epithelial Differentiation and Synergizes with Genistein to Enhance Transcriptional Response to Estrogen Receptor β Signaling

Mammary stromal adipocytes constitute an active site for the synthesis of the adipokine, adiponectin (APN) that may influence the mammary epithelial microenvironment. The relationship between "local," mammary tissue-derived APN and breast cancer risk is poorly understood. Here, we identify a novel mechanism of APN-mediated signaling that influences mammary epithelial cell proliferation, differentiation, and apoptosis to modify breast cancer risk. We demonstrate that early dietary exposure to soy protein isolate induced mammary tissue APN production without corresponding effects on systemic APN levels. In estrogen receptor (ER)-negative MCF-10A cells, recombinant APN promoted lobuloalveolar differentiation by inhibiting oncogenic signal transducer and activator of transcription 3 activity. In ER-positive HC11 cells, recombinant APN increased ERβ expression, inhibited cell proliferation, and induced apoptosis. Using the estrogen-responsive 4X-estrogen response element promoter-reporter construct to assess ER transactivation and small interfering RNA targeting of ERα and ERβ, we show that APN synergized with the soy phytoestrogen genistein to promote ERβ signaling in the presence of estrogen (17β-estradiol) and ERβ-specific agonist 2,3-bis(4-hydroxyphenyl)-propionitrile and to oppose ERα signaling in the presence of the ERα-specific agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol. The enhancement of ERβ signaling with APN + genistein cotreatments was associated with induction of apoptosis, increased expression of proapoptotic/prodifferentiation genes (Bad, p53, and Pten), and decreased antiapoptotic (Bcl2 and survivin) transcript levels. Our results suggest that mammary-derived APN can influence adjacent epithelial function by ER-dependent and ER-independent mechanisms that are consistent with reduction of breast cancer risk and suggest local APN induction by dietary factors as a targeted approach for promotion of breast health.

Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells

The risks and benefits of diets and supplements containing the estrogenic soy isoflavone genistein are not well established. We report that 10 nm genistein potently induces the granzyme B inhibitor, proteinase inhibitor 9 (PI-9) in MCF-7 human breast cancer cells. By inducing PI-9, genistein inhibits the ability of human natural killer (NK) cells to lyse the target breast cancer cells. In ERalphaHA cells, stably transfected MCF-7 cells, which contain elevated levels of estrogen receptor-alpha (ERalpha), 100 pm genistein or 17beta-estradiol potently induce PI-9 and prevent NK cells from killing the target breast cancer cells. The concentrations of genistein that fully induce PI-9 in MCF-7 cells, and in ERalphaHA cells, are far lower than those previously reported to elicit estrogenic responses through ERalpha. Because 4-hydroxytamoxifen, raloxifene, and ICI 182,780/Faslodex all block genistein induction of PI-9 and elevated levels of ERalpha enhance induction of PI-9, genistein acts via ERalpha to induce PI-9. Increasing levels of ERalpha in breast cancer cells results in a progressive increase in induction of PI-9 by genistein and in the cell's ability to evade killing by NK cells. Moderate levels of dietary genistein and soy flour effectively induce PI-9 in human breast cancers grown in ovariectomized athymic mice. A significant population consumes levels of genistein in soy products that may be high enough to induce PI-9, perhaps potentiating the survival of some preexisting breast cancers by enabling them to evade immunosurveillance.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and Na–K–Cl cotransporter NKCC1 isoform mediate the vasorelaxant action of genistein in isolated rat aorta

The soy phytoestrogen genistein is a potent vasorelaxant, but its mechanism of action is poorly understood. Here, we used endothelium-denuded rat aorta to investigate the role of the cyclic AMP(cAMP)-activated, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, and its associated Na–K–Cl cotransporter NKCC1. Isolated, endothelium-denuded rat aorta was contracted with phenylephrine 1 μM, and the vasorelaxant responses to genistein were investigated under conditions where CFTR was inhibited by DPC (diphenylamine-2-carboxylic acid) or glibenclamide ( n = 6 for compound). Both compounds fully antagonized the vasorelaxant responses to genistein, with IC 50 = 57 ± 18 μM and 42 ± 11 μM for DPC and glibenclamide respectively. H-89, a selective protein kinase A (PKA) inhibitor, blocked the vasorelaxant responses to genistein. Finally, the NKCC1 inhibitor, bumetanide fully antagonized the vasorelaxant responses to genistein against phenylephrine- or KCl-induced contractions, with IC 50 = 2.0 ± 0.2 μM and 1.6 ± 0.5 μM, respectively ( n = 6 for condition). These results strongly suggest that CFTR opening is involved in the vasorelaxant action of genistein, and that cAMP-dependent CFTR phosphorylation and chloride entry via the NKCC1 cotransporter are required for genistein action.

Estrogens modulate the gene expression of Wnt‐7a in cultured endometrial adenocarcinoma cells

The glycoprotein Wnt-7a participates in a signaling pathway that transmits information among uterine cell types. Disruption of this pathway by the transplacentally acting carcinogen diethylstilbestrol (DES) is associated with morphological abnormalities of the female reproductive tract (FRT). This raises the question whether estrogens in the diet might also interfere with this pathway. Therefore, this study investigated the influence of the steroid hormone 17beta-estradiol (E2), the mycotoxin zearalenone (ZEN), the soy phytoestrogen genistein (GEN), and DES on the expression of Wnt-7a in an endometrial adenocarcinoma cell line (Ishikawa cells) by reverse transcription/competitive PCR. In addition, the enzymatic activity of alkaline phosphatase (ALP) was determined, which is estrogen receptor (ER)-dependently regulated in Ishikawa cells. After treatment of Ishikawa cells with E2, ZEN, GEN, and DES, a decrease in the gene expression of Wnt-7a was observed. Maximum effect (50% reduction) was observed after treatment with concentrations that induced maximum expression of the ALP. Experiments in the presence of the ER antagonist (ICI 182,780) suggested that the ER is involved in the regulation of Wnt-7a in Ishikawa cells. In conclusion, interference with the expression of Wnt genes in the FRT might be a novel mechanism by which estrogens disrupt the function of the FRT.

Children’s Health: Sour News for Soy Formula?

Naturally occurring phytoestrogens have been intensively studied for health effects in adults. However, studies of soy formula, which delivers high levels of phytoestrogens to infants, have not extended much beyond ensuring that babies are growing and developing normally. Still, soy formula has been considered a safe alternative to milk-based formulas for some 40 years. Recent studies from the University of Illinois at Urbana–Champaign now show that the soy phytoestrogen genistein can alter intestinal cell proliferation and migration, with unknown effects for infants fed soy formula. “We are feeding infants soy formula as their sole source of nutrition for the first four to six months of life, a period of time when many systems are immature and undergoing development,” says Sharon Donovan, a professor of nutrition involved in both studies. It is known that infants metabolize genistein and can have some circulating level of the bioactive form. But whether the effects are good, bad, or even measurable is unknown and fiercely debated. “Why [soy formula] has not received more research attention, I’m not sure,” says Retha Newbold, an NIEHS toxicologist who has investigated the developmental effects of genistein and other estrogenic substances for more than 25 years. In the first Illinois study, published in the June 2004 Journal of Nutrition, researchers exposed human intestinal cells to varying doses of genistein and noted effects on cell numbers, DNA replication, apoptosis, and cell cycle. At low doses, genistein acted as a weak estrogen and stimulated cell growth; at high doses, the compound inhibited proliferation and altered cell cycle dynamics. This biphasic response correlates with how genistein is thought to exert its effects. In the other study, published in the February 2005 Pediatric Research, 24 2-day-old piglets were divided into three dietary groups for eight days, receiving plain sow milk replacer or replacer with either a low or high dose of genistein. The high-dose piglets had circulating concentrations of genistein on par with those of soy formula–fed infants. At 10 days of age, there were no significant differences in weight gain, intestinal length or growth, nutrient uptake, or digestive enzyme activity among the piglets. However, there was a 50% decrease in intestinal cell proliferation and a 20% decrease in cell migration associated with the high genistein dose. Donovan cautions that it’s premature to draw conclusions about negative or positive effects of infant soy formula. “This is what we see when we look at genistein alone,” she says, “but what happens when you look at a whole soy formula?” More than 20 million American infants have been fed soy formula in the last 25 years, and their growth and development have been equal to that of infants fed milk-based formula, according to Thomas Badger, director and senior investigator at the Arkansas Children’s Nutrition Center in Little Rock. “If there have been no problems in more than twenty million people exposed to soy formula, then there is no human evidence of a problem,” says Badger. Still, many researchers believe that more information is needed about the safety of infant soy formula. Phytoestrogen doses comparable to what infants receive through soy formula have been shown to cause cancer in some animal studies if given before puberty. “I think too little is known to conclude that soy formula is safe for the general infant population,” says Newbold.

Soy-induced brain atrophy?

Epidemiological research has demonstrated a positive correlation between tofu consumption and brain atrophy in men. Because correlation does not prove causation, correlation-based hypotheses should be tested against the availability of possible causal mechanisms. While it has been shown that the soy phytoestrogen genistein inhibits neuroprotective functions in cell cultures, recent in-vivo findings strengthen the case for a possible causal mechanism of soy-induced neurodegeneration. The author suggests possible responses to this data regarding soy consumption.

Peroxisome Proliferator-activated Receptor γ (PPARγ) as a Molecular Target for the Soy Phytoestrogen Genistein

The principal soy phytoestrogen genistein has an array of biological actions. It binds to estrogen receptor (ER) alpha and beta and has ER-mediated estrogenic effects. In addition, it has antiestrogenic effects as well as non-ER-mediated effects such as inhibition of tyrosine kinase. Because of its complex biological actions, the molecular mechanisms of action of genistein are poorly understood. Here we show that genistein dose-dependently increases estrogenic transcriptional activity in mesenchymal progenitor cells, but its biological effects on osteogenesis and adipogenesis are different. At low concentrations (< or =1 microm), genistein acts as estrogen, stimulating osteogenesis and inhibiting adipogenesis. At high concentrations (>1 microm), however, genistein acts as a ligand of PPARgamma, leading to up-regulation of adipogenesis and down-regulation of osteogenesis. Transfection experiments show that activation of PPARgamma by genistein at the micromolar concentrations down-regulates its estrogenic transcriptional activity, while activation of ERalpha or ERbeta by genistein down-regulates PPARgamma transcriptional activity. Genistein concurrently activates two different transcriptional factors, ERs and PPARgamma, which have opposite effects on osteogenesis or adipogenesis. As a result, the balance between activated ERs and PPARgamma determines the biological effects of genistein on osteogenesis and adipogenesis. Our findings may explain distinct effects of genistein in different tissues.

Differential Effects of Genistein on Cell Proliferation, Cyclin B1, and p34cdc2 in Transformed and Nontransformed Human Breast Cells

Phytoestrogens present in legumes and other plants have been the subject of considerable attention due to their reported beneficial impact on chronic diseases, including breast cancer. Recently, it has been reported that normal human mammary epithelial cells may be more sensitive than neoplastic human breast cells to the antiproliferative action of the soy phytoestrogen genistein. The present studies were conducted in order to characterize cell cycle-associated events responsible for the differential sensitivity to genistein between nontransformed and transformed human breast cells. Genistein was more effective in inhibiting proliferation of nonneoplastic MCF-10F cells (IC50 ~19‐22 mM) compared to neoplastic MCF-7 cells (IC50 ~45‐55 mM). In addition, exposure of MCF-10F cells to genistein caused a reversible G2/M block in cell cycle progression, in contrast to that for MCF-7. The G2/M block in MCF-10F was associated with significant increases in the content of two proteins, cyclin B1 (2-fold) and the phosphorylated form of the cyclin-dependent kinase p34 cdc2 (3.7-fold). No such changes in these proteins were observed in MCF-7 cells treated with similar concentrations of genistein. This finding of differential sensitivity to genistein between transformed MCF-7 cells and nontransformed MCF-10F breast cells suggests that genistein and related flavonoids could be useful as chemopreventive agents targeting earlier stages in the process of neoplastic transformation of human breast cells.

Actions of the soy phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growth factor β

The structural similarity, but non-identity, between 17β-oestradiol and the soy phytoestrogen genistein suggests that the two compounds will have actions that may be identical in some target biological systems, but different in others. Epidermal growth factor (EGF)-stimulated proliferation of human mammary epithelial cells (that do not express the oestrogen receptor) was significantly suppressed at genistein concentrations (5–10μM) that are attainable physiologically. Others have shown previously that transforming growth factor β (TGFβ) has similar growth-inhibitory effects on human cells. Analysis of the conditioned medium of human mammary epithelial cells exposed to genistein plus EGF showed increased levels of TGFβ relative to those in the medium of cells exposed to EGF or genistein alone. Related experiments in a primate model of menopause demonstrated that ingestion of soy containing isoflavones was correlated with the suppression of neurodegeneration-relevant phosphorylation of the microtubule-associated protein tau, while intake of Premarin (a hormone replacement therapy that is commonly prescribed for women) was not correlated. The results discussed here indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFβ activity.

Actions of the soy phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growth factor beta.

The structural similarity, but non-identity, between 17beta-oestradiol and the soy phytoestrogen genistein suggests that the two compounds will have actions that may be identical in some target biological systems, but different in others. Epidermal growth factor (EGF)-stimulated proliferation of human mammary epithelial cells (that do not express the oestrogen receptor) was significantly suppressed at genistein concentrations (5-10 microM) that are attainable physiologically. Others have shown previously that transforming growth factor beta (TGFbeta) has similar growth-inhibitory effects on human cells. Analysis of the conditioned medium of human mammary epithelial cells exposed to genistein plus EGF showed increased levels of TGFbeta relative to those in the medium of cells exposed to EGF or genistein alone. Related experiments in a primate model of menopause demonstrated that ingestion of soy containing isoflavones was correlated with the suppression of neurodegeneration-relevant phosphorylation of the microtubule-associated protein tau, while intake of Premarin (a hormone replacement therapy that is commonly prescribed for women) was not correlated. The results discussed here indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFbeta activity.

Soy phytoestrogens decrease the estrogenic activity of estradiol in an in vitro bioassay using a human endometrial cell line

Previously, soy phytoestrogens (SPEs) were considered to be merely weak estrogens because their binding affinity for the estrogen receptor (ER) was considerably less than that of estradiol. Currently, with our recent understanding of differences in ERα and ERβ, that view must be revised. Some phytoestrogens have a high affinity, relative to estradiol, for ERβ. The soy phytoestrogen genistein, for example, has a 20-fold greater affinity for the ERβ relative to ERα. Given the different tissue distribution of the α and β forms of ER, there is a cellular basis for tissue-selective effects of genistein.