Phytoestrogen Genistein Research Articles

Genistein Stimulates Jejunum Chloride Secretion via an Akt-Mediated Pathway in Intact Female Mice

Background/Aims: We have previously shown that daily subcutaneous injections with the naturally occurring phytoestrogen genistein (600 mg genistein/kg body weight/day, 600G) results in a significantly increased basal intestinal chloride, Cl^-, secretion (I_sc, a measure of transepithelial secretion) in intact C57BL/6J female mice after 1-week of treatment, compared to controls (DMSO vehicle injected). Removal of endogenous estrogen via ovariectomy (OVX) had no effect on the 600G-mediated increase in basal I_sc. Methods: Given the estrogen-like characteristics of genistein, we compared the effects of daily estradiol (E2) injections (10 mg E2/kg body weight/day, 10E2) on basal I_sc in intact and OVX mice. In intact mice, 10E2 was without effect on basal I_sc, however, in OVX mice, 10E2 significantly increased basal I_sc (mimicked 600G). The goal of the current study was to characterize the intracellular signaling pathways responsible for mediating 600G- or 10E2-stimulated increases in basal I_sc in intact female or OVX mice. Results: We measured total protein expression in isolated segments of jejunum using western blot from the following six groups of mice; intact or OVX with; 600G, 10E2 or control. The proteins of interest were: Akt, p-Akt, p-PDK1, p-PTEN, p-c-Raf, p-GSK-3β, rap-1 and ERK1/2. All blots were normalized to GAPDH levels (n = 6-18/group). Conclusion: These data suggest that the presence of the endogenous sex steroid, estrogen, modifies the intracellular signaling pathway required to mediate Cl^- secretion when the intestine is exposed to exogenous 600G or E2. These studies may have relevance for designing pharmacological tools for women with intestinal chloride secretory dysfunctions.

Multi-walled carbon nanotubes for the dispersive solid-phase extraction of the phytoestrogen genistein from water samples using high performance liquid chromatography

Diagram of the mechanism of GEN adsorption on MWCNTs.

Genistein disrupts glucocorticoid receptor signaling in human uterine endometrial Ishikawa cells.

Background: The link between environmental estrogen exposure and defects in the female reproductive tract is well established. The phytoestrogen genistein is able to modulate uterine estrogen receptor (ER) activity, and dietary exposure is associated with uterine pathologies. Regulation of stress and immune functions by the glucocorticoid receptor (GR) is also an integral part of maintaining reproductive tract function; disruption of GR signaling by genistein may also have a role in the adverse effects of genistein.Objective: We evaluated the transcriptional response to genistein in Ishikawa cells and investigated the effects of genistein on GR-mediated target genes.Methods: We used Ishikawa cells as a model system to identify novel targets of genistein and the synthetic glucocorticoid dexamethasone through whole genome microarray analysis. Common gene targets were defined and response patterns verified by quantitative real-time reverse-transcription polymerase chain reaction. The mechanism of transcriptional antagonism was determined for select genes.Results: Genistein regulated numerous genes in Ishikawa cells independently of estradiol, and the response to coadministration of genistein and dexamethasone was unique compared with the response to either estradiol or dexamethasone alone. Furthermore, genistein altered glucocorticoid regulation of GR target genes. In a select set of genes, co-regulation by dexamethasone and genistein was found to require both GR and ERα signaling, respectively.Conclusions: Using Ishikawa cells, we observed that exposure to genistein resulted in distinct changes in gene expression and unique differences in the GR transcriptome.Citation: Whirledge S, Senbanjo LT, Cidlowski JA. 2015. Genistein disrupts glucocorticoid receptor signaling in human uterine endometrial Ishikawa cells. Environ Health Perspect 123:80–87; http://dx.doi.org/10.1289/ehp.1408437

Effect of genistein on regenerative angiogenesis using zebrafish as model organism

We examined the effects of phytoestrogen genistein on zebrafish caudal fin regeneration and found that genistein could inhibit fin generation in μM concentration. Fish were injected with varying concentrations (10–100μM) of genistein for 5 days after caudal fin amputation, and regeneration of the fin was evaluated by analyzing caudal fin length and newly formed vascular region. Regeneration of amputated fins was inhibited or delayed to the maximum of 64.7% in dorsal region, 63.35% in cleft region and 66.54% in ventral region after genistein treatment unlike the control that completely regenerated their fins after 5 days post-amputation (DPA). PCR data showed a clear reduction in vascular endothelial growth factor (VEGF) expression on exposure to genistein while a complete inhibition was observed with sunitinib (SU 11652) an inhibitor of VEGF signaling. The ability of genistein to inhibit regenerative angiogenesis of caudal fin probably by down regulating VEGF, the key player of angiogenesis and the results obtained with SU 11652 is suggestive of the involvement of VEGF signaling during regeneration. These results demonstrate that zebrafish could be a good model in elucidating molecular mechanisms that are responsible for fin regeneration.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phytoestrogen genistein on the activity and the presence of steroidogenic enzyme proteins in cultured granulosa cells of pigs

Environmental estrogens such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) and phytoestrogens (e.g. genistein; G) are known to influence endocrine and reproductive processes in humans and animals. Because living organisms are usually exposed to small, non toxic, doses of dioxins and phytoestrogens, the aims of the study were to determine the effects of small, environmentally relevant doses of TCDD (100pM) and/or genistein (500nM) on: (1) the activity of steroidogenic enzymes (cholesterol side-chain cleavage enzyme, P450scc; 3β-hydroxysteroid dehydrogenase, 3β-HSD and aromatase, P450arom) and (2) amount of protein of the enzymes in granulosa cells isolated from medium and large ovarian follicles of pigs. To determine the activity of the enzymes, the incubation medium was supplemented with specific steroid substrates (25-hydroxycholesterol; pregnenolone; testosterone) of particular steroidogenic enzymes (P450scc, 3β-HSD and P450arom, respectively). Subsequently, the production of progesterone (P450scc and 3β-HSD) or estradiol (P450arom) was compared in the presence and absence of the appropriate steroid precursor. Neither genistein nor genistein combined with TCDD affected activity of P450arom and relative amounts of steroidogenic enzyme proteins in the examined granulosa cells of pigs. In contrast, genistein alone and in combination with TCDD decreased P450scc and 3β-HSD activity as well as progesterone production in granulosa cells isolated from medium and large follicles of pigs. Because TCDD alone did not affect steroid hormone production or enzyme activity, the above effects should be ascribed solely to genistein. It appears that the effects of the examined doses of TCDD and genistein on granulosal cell functions were not additive.

Dietary exposure in utero and during lactation to a mixture of genistein and an anti-androgen fungicide in a rat mammary carcinogenesis model

Endocrine disruptors may play substantial roles in the high incidence of breast cancer. We previously described how early exposure to the mixture of phytoestrogen genistein (G) and the anti-androgen vinclozolin (V) affects peripubertal mammary development. This study evaluates the carcinogenic potential of exposure to V alone or associated with G from conception until weaning in Wistar rats. Dams were exposed to V, G or GV during pregnancy/lactation. At PND50 offspring were treated with DMBA[7,12-dimethylbenz(a)anthracene]. V or GV maternal exposure decreased number of DMBA-induced mammary tumors in the offspring, without significant modifications in tumor incidence, multiplicity and latency. G exposure decreased number of tumors, incidence and multiplicity. Unexpectedly, GV exposure increased tumor volume (p=0.04 vs controls) and epithelial proliferation (p=0.001 vs controls; p=0.005 vs G,V only). All tumors were in situ carcinomas. Concluding, maternal gestation/lactation exposure to a vinclozolin and genistein mixture significantly increases offspring tumor growth without changes in carcinogenesis susceptibility.

In vitro and in vivo effects of phytoestrogens on protein turnover in rainbow trout (Oncorhynchus mykiss) white muscle

Soybeans and other legumes investigated as fishmeal replacements in aquafeeds contain phytoestrogens capable of binding to and activating estrogen receptors. Estradiol has catabolic effects in salmonid white muscle, partially through increases in protein turnover. The current study determines whether phytoestrogens promote similar effects. In rainbow trout (Oncorhynchus mykiss) primary myocyte cultures, the phytoestrogens genistein, daidzein, glycitein, and R- and S-equol reduced rates of protein synthesis and genistein, the phytoestrogen of greatest abundance in soy, also increased rates of protein degradation. Increased expression of the ubiquitin ligase fbxo32 and autophagy-related genes was observed with high concentrations of genistein (100μM), and R- and S-equol (100μM) also up-regulated autophagy-related genes. In contrast, low genistein concentrations in vitro (0.01–0.10μM) and in vivo (5μg/g body mass) decreased fbxo32 expression, suggesting a potential metabolic benefit for low levels of genistein exposure. Phytoestrogens reduced cell proliferation, indicating that effects of phytoestrogens extend from metabolic to mitogenic processes. Co-incubation of genistein with the estrogen receptor (ER) antagonist, ICI 182,780, ameliorated effects of genistein on protein degradation, but not protein synthesis or cell proliferation, indicating that effects of genistein are mediated through ER-dependent and ER-independent mechanisms. Collectively, these data warrant additional studies to determine the extent to which dietary phytoestrogens, especially genistein, affect physiological processes that impact growth and nutrient retention.

Genistein Protects Female Nonobese Diabetic Mice from Developing Type 1 Diabetes When Fed a Soy- and Alfalfa-free Diet

The objective of this study was to determine the effects of the phytoestrogen genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes (T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage once every day for up to 180 days. Five groups of mice (approximately 24 animals/group; 6-7 weeks of age) were included: naive control, vehicle control (25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20 mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the study and were monitored for blood glucose changes every week. When compared to the vehicle control, exposure to 2-mg/kg GEN produced significant decreases ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250 mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the study. However, during the later stages of the study (i.e., after week 23), the 2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were observed starting at week 16, while the incidence of severe diabetes was significantly decreased with the changes being observed initially at weeks 18 and 17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines of evidence, including histopathological analysis, suggested that GEN protected the pancreas from autoimmune destruction. However, this protective effect of GEN was absent when female NOD mice were maintained on NTP-2000 rodent diet, which contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN reduced the incidence and increased the time to onset of T1D in female NOD mice but only when fed a soy- and alfalfa-free diet.

Soybean phytoestrogen enhances the antimicrobial peptides beta‐defensin‐2 synthesis in endometrial epithelial cells with lipopolysaccharides and polyinosinic‐polycytidylic acid stimulation (639.8)

β‐defensins (BD) are antimicrobial peptides that contribute the mucosal innate immunity. Its expression is regulated by sex steroid hormones. Phytoestrogens genistein (Ge) and daidzein (Di) mostly found in soybean are structurally similar to estrogen. This study aims to examine the effect of phytoestrogens on the synthesis and secretion of BD in endometrial epithelial cells under pathogenic conditions. The cells were cultivated in estrogen‐free media in the presence of phytoestrogen for 48 h following with lipopolysaccharide (LPS) or polyinosinic‐polycytidylic acid (poly I:C) stimulation. The mRNA expression and the secretion of BD were analyzed by real‐time PCR and ELISA, respectively. The cells expressed and secreted BD2 with a greater extent than other BD subtypes (BD1, 3 or 4). A 4‐h inoculation with poly I:C decreased the BD2 secretion. Pretreatment with Ge (0.1 nM or 1 μM) increased both the expression and secretion of BD2 in response to LPS. However, Ge at 0.1 nM prevented the poly I:C suppressed BD2 secretion. Under poly I:C inoculation, Di (0.1 nM) induced the BD2 secretion but it did not affect the expression. This study provides the first evidence and a role of BD in mucosal defense against pathogen in glandular endometrial epithelium. The differential modulation of the expression and secretion of BD by soybean phytoestrogen could be applied for protection of female reproductive tract from pathogen invasions.Grant Funding Source: Thailand Research Fund

Comparative Effects of Soy Phytoestrogens and 17β-Estradiol on DNA Methylation of a Panel of 24 Genes in Prostate Cancer Cell Lines

Major phytoestrogens genistein and daidzein have been reported to have the ability to reverse DNA methylation in cancer cell lines. The mechanism by which genistein and daidzein have an inhibiting action on DNA methylation is not well understood. The aim of this study was to investigate the effects of soy phytoestrogens and the natural estrogen 17β-estradiol (E2) to determine whether one of the estrogen receptors is mobilized for the action of these compounds on DNA methylation. We also made a comparative study with a DNA methylation inhibitor (5-azacytidine) and a DNA methylation activator (budesonide). Three prostate cell lines, PC-3, DU-145, and LNCaP, were treated with 40 μM genistein, 110 μM daidzein, 2 μM budesonide, 2 μM 5-azacytidine, and 10 μM E2. In these 3 human prostate cancer cell lines, we performed methylation quantification using methyl-profiler-DNA-methylation analysis. Soy phytoestrogens and E2 induced a demethylation of all the promoter regions studied except for those that were unmethylated in control cells. Our results showed that E2 induces, like soy phytoestrogen, a decrease in DNA methylation in prostate cancer cell lines. This action may be mediated through ERβ.

The Effects of Different Antioxidants on the Activity of Cerebrocortical MnSOD and Na,K-ATPase from post mortem Alzheimer’s Disease and Age-matched Normal Brains

Among the markers and targets of the early phase of Alzheimer's disease (AD) pathogenesis MnSOD (mitochondrial dysfunction) and Na-pump (disturbances in function/regulation) are often highlighted. This paper focused on comparison of the effects of three antioxidants on the activity of cerebrocortical MnSOD and Na,K-ATPase from post mortem Alzheimer's disease and age-matched normal brains. Antioxidant compounds with different origins: natural glutathione, synthetic UPF peptides (glutathione analogues) and phytoestrogen genistein were investigated. Firstly, MnSOD and Na,K-ATPase activities were found to be decreased in the post mortem AD brains compared with age-matched controls. Secondly, GSH had no effect on MnSOD activity, but decreased Na,K-ATPase activity both in the control and AD brains. Thirdly, UPF1 and UPF17 increased MnSOD activity, and UPF17 suppressed Na,K-ATPase activity. Further studies are needed to clarify, if the inhibitory effect of UPF17 on Na,K-ATPase could abolish the beneficial effect gained from MnSOD activation. Both the antioxidative potential of genistein and its potency to up-regulate Na,K-ATPase activity make it an attractive candidate substance to suppress the early phase of the pathogenesis of AD.

Exposure to Environmentally Relevant Concentrations of Genistein during Activation Does Not Affect Sperm Motility in the Fighting FishBetta splendens

Sperm collected from male fighting fish Betta splendens were activated in control water, water containing the ion-channel blocker gadolinium (a putative positive control), or water containing the isoflavone phytoestrogen genistein to determine the effects of acute genistein exposure on male reproductive function. Computer-assisted sperm analysis was used to quantify the proportion of sperm that were motile and the swimming velocity of those sperm. The highest concentration of gadolinium (100 μM) tested was effective at reducing sperm motility and velocity, but neither concentration of genistein tested (3.7 nM or 3.7 μM) significantly affected these sperm parameters. Our findings suggest that acute exposure to waterborne phytoestrogens during activation does not reduce the motility of fish sperm.

Genistein effects on quality of life and depression symptoms in osteopenic postmenopausal women: a 2-year randomized, double-blind, controlled study

Postmenopausal estrogen decline is implicated in several age-related physical and psychological changes in women, including decreases in perceived quality of life. The phytoestrogen genistein at a dose of 54 mg daily in osteopenic postmenopausal women after 2 years implies an improvement on quality of life and depression symptoms. Postmenopausal estrogen decline is implicated in several age-related physical and psychological changes in women, including decreases in perceived quality of life (QoL). A number of trials with hormone therapy showed beneficial effects of the intervention on quality of life parameters. However, because of known or suspected serious side effects of conventional hormone therapy, there is a need for alternatives. We conducted a double-blind randomized placebo-controlled trial using the isoflavone genistein, 54 mg, or placebo for 2 years. In this trial, we recruited 262 postmenopausal women aged 49 to 67 years. At baseline, after 1 year, and at final visit, participants filled in the Short Form of 36 questions (SF-36) and the Zung Self-rating Depression Scale (ZSDS). For the placebo group, scores on all dimensions of the SF-36 decreased after 1 and 2 years. The genistein group showed increases on all dimensions of the SF-36 at the end of the study. There were, however, statistically significant differences in changes of scores between the two intervention groups. For the ZSDS, similarly, significant differences were found between groups. In conclusion, the findings of this randomized trial showed that genistein improves quality of life (health status, life satisfaction, and depression) in osteopenic postmenopausal women.

In silico evidences for the binding of phthalates onto human estrogen receptor α, β subtypes and human estrogen-related receptor γ

Being lipophilic xenobiotic chemicals, phthalates from the surrounding environments can easily be absorbed into the biological system, thereby causing various health problems including cancer and endocrine disruption in test animals and also in humans. In the present in silico study employing Glide, Schrödinger Suite 2012, we analysed in detail the binding affinities of 12 commonly used diphthalates and their metabolites (corresponding mono ester and phthalic acid) onto the ligand-binding domain (LBD) of the human estrogen receptor α (hERα), human estrogen receptor β (hERβ) and human estrogen related receptor γ (hERRγ). Natural ligand 17β estradiol (E2), known xenoestrogen bisphenol A, the phytoestrogen genistein, the agonists/antagonists 4-hydroxy tamoxifen and raloxifene were also docked onto these receptors as positive controls for comparing the binding efficiencies with that of phthalates and their metabolites. Results revealed that E2 had less binding affinity to the receptors in comparison to certain phthalates, i.e. maximum binding scores (G score, kcal/mol) were diisononyl phthalate ( − 9.44) to hERα, monophenyl phthalate ( − 8.66) to hERβ and di(2-ethylhexyl)phthalate ( − 9.38) to hERRγ. The most concerned monophthalates established additional H bonds with certain surrounding crucial amino acid residues in the LBD, and thus showed more affinity to all the receptors than even the natural ligand and other well-characterised xenoestrogens as demonstrated in this study. Briefly, this study gives an insight into the virtual binding behaviours of commonly used phthalates and their metabolites onto hERs and hERRγ, which would accelerate further in vitro mechanistic, preclinical and clinical studies on real in vitro or in vivo platforms.

Comparative study of dietary soy phytoestrogens genistein and equol effects on growth parameters and ovarian development in farmed female beluga sturgeon, Huso huso

Oocyte maturation in fish is a hormonally regulated process. In the light of long-term oocyte maturation in beluga, the aim of this research was to study the estrogenic effects of different concentrations of soy dietary genistein (GE) and equol (EQ) on the growth performance and ovary development in farmed female Huso huso. Fish were fed with concentrations 0 (control), 0.2, 0.4, 0.8 and 1.6 g of EQ and GE per kg of isoproteic (CP 45 %) and isoenergetic (19.5 MJ/kg) diets during a year. Blood samples and ovary biopsies were collected from each fish seasonally. The main results of the present experimentation are that growth performance was not affected significantly both in GE and EQ (P > 0.05). EQ at concentration 0.4 g/kg had more estrogenic effects than other concentrations of EQ and GE in beluga so that 64 % of fish were matured sexually. Some reproductive indices such as oocyte diameter, testosterone (T) and 17β-estradiol (E₂) increased significantly at EQ 0.4 g/kg at the end of experiment (P < 0.05), while 17α-hydroxy progesterone level (17α-OHP) showed no significant changes at all concentrations. Biochemical indices such as calcium, phosphorous and cholesterol increased at GE concentrations, but decreased at EQ concentrations similarly at the end of experiment. There was a negative relationship between plasma phosphorous and alkaline phosphatase enzyme levels. Based on results, EQ at concentration 0.4 g/kg improved oocyte development more than the other concentrations of GE and EQ, and therefore, it can be used as an additive to diets for inducing ovary development in this species.

AB1029 Genistein prevents quality of life osteopenic post-menopausal womens: A 2-year randomized, double-blind, controlled-study

BackgroundPostmenopausal estrogen decline is implicated in several age-related physical and psychological changes in women, including decreases in perceived quality of life (QoL). A number of trials with hormone therapy showed...

Genistein Excitation of Gonadotrophin‐Releasing Hormone Neurones in Juvenile Female Mice

We investigated the effects of the phytoestrogen genistein on gonadotrophin-releasing hormone (GnRH) neurones using single-cell electrophysiology on GnRH-green fluorescent protein (GFP) transgenic juvenile female mice. Perforated patch-clamp recordings from GnRH-GFP neurones showed that approximately 83% of GnRH neurones responded to 30 μm genistein with a markedly prolonged membrane depolarisation. This effect not only persisted in the presence of tetrodotoxin, but also in the presence of amino acid receptor antagonists, indicating the direct site of action on postsynaptic GnRH neurones. Using a voltage clamp technique, we found that 30 μm genistein increased the frequency of synaptic current of GnRH neurones clamped at -60 mV in the presence of glutamate receptor blocker but not GABAA receptor blocker. Pre-incubation of GnRH neurones with 30 μm genistein enhanced kisspeptin-induced membrane depolarisation and firing. GnRH neurones of juvenile mice injected with genistein in vivo showed an enhanced kisspeptin response compared to vehicle-injected controls. The transient receptor potential channel (TRPC) blocker 2-aminoethoxydiphenyl borate (75 μm) blocked the genistein-mediated response on GnRH neurones. These results demonstrate that genistein acts on GnRH neurones in juvenile female mice to induce excitation via GABA neurotransmission and TRPCs to enhance kisspeptin-induced activation.

The phytoestrogen Genistein inhibits the anti-tumor activity of cisplatin in MCF-7 breast and HT29 colon cancer cells

The phytoestrogen Genistein inhibits the anti-tumor activity of cisplatin in MCF-7 breast and HT29 colon cancer cells

Review: Mammary development in swine: effects of hormonal status, nutrition and management

Farmer, C. 2013. Review: Mammary development in swine: effects of hormonal status, nutrition and management. Can. J. Anim. Sci. 93: 1-7. There are three phases of rapid mammary accretion in swine, namely, from 90 d of age until puberty, during the last third of gestation and throughout lactation. Nutrition, endocrine status and management of gilts or sows during those periods can affect mammary development. More specifically, in growing gilts, feed restriction as of 90 d of age hinders mammary development and either supplying the phytoestrogen genistein or increasing circulating concentrations of prolactin stimulates mammogenesis. In late gestation, inhibition of relaxin or prolactin drastically diminishes mammary development and overly increasing dietary energy has a detrimental effect on mammogenesis. It also appears that feeding of the gestating sow can affect the mammary development of her offspring once it reaches puberty. Various management factors such as litter size, nursing intensity and use or n...

Review: Mammary development in swine: effects of hormonal status, nutrition and management

Farmer, C. 2013. Review: Mammary development in swine: effects of hormonal status, nutrition and management. Can. J. Anim. Sci. 93: 1–7. There are three phases of rapid mammary accretion in swine, namely, from 90 d of age until puberty, during the last third of gestation and throughout lactation. Nutrition, endocrine status and management of gilts or sows during those periods can affect mammary development. More specifically, in growing gilts, feed restriction as of 90 d of age hinders mammary development and either supplying the phytoestrogen genistein or increasing circulating concentrations of prolactin stimulates mammogenesis. In late gestation, inhibition of relaxin or prolactin drastically diminishes mammary development and overly increasing dietary energy has a detrimental effect on mammogenesis. It also appears that feeding of the gestating sow can affect the mammary development of her offspring once it reaches puberty. Various management factors such as litter size, nursing intensity and use or non-use of a teat in the previous lactation will affect the amount of mammary tissue present at the end of lactation. Mammary development is followed by the essential process of involution whereby a rapid and drastic regression in parenchymal tissue takes place. It can occur either after weaning or in early lactation when teats are not being regularly suckled. Despite our current knowledge, much remains to be learned in order to develop the best management strategies for replacement gilts, and gestating and lactating sows that will maximize their milk production.