Hepatotoxic effect of dietary phytoestrogens on juvenile cultured Russian sturgeon (Acipenser gueldenstaedtii).

In the last two decades, much controversy has grown over the use of soybean products in aquafeeds, especially for carnivorous fish like sturgeons. One point of discussion is the effect of soybean phytoestrogens on fish health. There are many aspects of phytoestrogen utilization in aquafeeds, therefore, the aim of this study is to verify if common legume phytoestrogens can affect juvenile cultured sturgeon erythrocyte and hepatocyte genotoxicity and cause liver pathology. Russian sturgeons were fed from 100 till 365 dph1 with daidzein, genistein, and coumestrol supplemented diets in concentrations: 10, 0.05 and 0.001 g kg-1 of feed, respectively. The SCGE2 method combined with qPCR of three genes involved in DNA repair and genome maintenance, namely cyp1a1, gaad45a and p53 were analyzed. The results were compared with histopathological evaluation of liver tissue. In fish fed with coumestrol supplemented diet, DNA strand damage was the highest in both erythrocytes and hepatocytes, however, simultaneously the lowest level of oxidative DNA damage was found. Additionally, slightly elevated expression of the p53 gene was observed along with a decreased number of apoptotic hepatocytes, which suggests that low concentration of coumestrol may support DNA repair mechanisms in the liver. Although, daidzein showed a preventive effect only against fibrosis. Isoflavones did not show a significant effect on DNA damage in studied cells. Genistein was found to increase macro- and microvesicular steatosis, portal hepatitis and fibrosis, indicating its negative role in the development of liver injuries. Daidzein alleviated some sturgeon liver damage, especially macrovesicular steatosis and interface hepatitis. However, it increased hepatocyte apoptosis, which may suggest daidzein potentially inducing liver injury, though not manifested by other histopathological lesions. Therefore, it can be concluded that at given concentrations, the tested phytoestrogens did not show clearly hepatoprotective effect in sturgeons.

Dietary phytoestrogen diosgenin interrupts metabolism, physiology, and reproduction of Swiss albino mice: Possible mode of action as an emerging environmental contaminant, endocrine disruptor and reproductive toxicant.

Dietary phytoestrogens are the main source of environmental contamination due to their estrogen-mimicking and endocrine-disrupting effects, posing a threat to microbial, soil, plant, and animal health. Diosgenin, a phytosteroid saponin, is used in many traditional medicines, nutraceuticals, dietary supplements, contraceptives, and hormone replacement therapies against numerous diseases and disorders. It is important to be aware of the potential risks associated with diosgenin, as well as its potential to cause reproductive and endocrine toxicity. Due to the lack of research on the safety and probable adverse side effects of diosgenin, this work evaluated the endocrine-disrupting and reproductive toxicity of diosgenin in albino mice by following acute toxicity (OECD-423), repeated dose 90-day oral toxicity (OECD-468), and F1 extended one-generation reproductive toxicity (OECD-443) studies. Diosgenin was found to be slightly toxic, with LD50 for male and female mice being 546.26 and 538.72 mg/kg, respectively. Chronic exposure of diosgenin (10, 50, 100, and 200 mg/kg) generated oxidative stress, depleted antioxidant enzymes, disturbed homeostasis of the reproductive hormones, and interrupted steroidogenesis, germ cell apoptosis, gametogenesis, sperm quality, estrous cycle, and reproductive performance in the F0 and F1 offspring. Long-term oral exposure of diosgenin to the mice disturbed the endocrine and reproductive functions and generated transgenerational reproductive toxic effects in F0 and F1 offspring. These results suggest that diosgenin should be used carefully in food products and medical applications due to its potential endocrine-disrupting and reproductive toxic effects. The findings of this study provide a better understanding of the potential adverse effects of diosgenin and the need for appropriate risk assessment and management of its use.

Estrogenic in vitro evaluation of zearalenone and its phase I and II metabolites in combination with soy isoflavones.

Humans and animals are exposed to multiple substances in their food and feed that might have a negative health impact. Among these substances, the Fusarium mycoestrogen zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL) and α-zearalanol (α-ZAL) are known to possess endocrine disruptive properties. In a mixed diet or especially animal feed, these potential contaminants might be ingested together with naturally occurring phytoestrogens such as soy isoflavones. So far, risk assessment of potential endocrine disruptors is usually based on adverse effects of single compounds whereas studies investigating combinatorial effects are scarce. In the present study, we investigated the estrogenic potential of mycoestrogens and the isoflavones genistein (GEN), daidzein (DAI) and glycitein (GLY) as well as equol (EQ), the gut microbial metabolite of DAI, in vitro alone or in combination, using the alkaline phosphatase (ALP) assay in Ishikawa cells. In the case of mycoestrogens, the tested concentration range included 0.001 to 10 nM with multiplication steps of 10 in between, while for the isoflavones 1000 times higher concentrations were investigated. For the individual substances the following order of estrogenicity was obtained: α-ZEL > α-ZAL > ZEN > GEN > EQ > DAI > GLY. Most combinations of isoflavones with mycoestrogens enhanced the estrogenic response in the investigated concentrations. Especially lower concentrations of ZEN, α-ZEL and α-ZAL (0.001-0.01 nM) in combination with low concentrations of GEN, DAI and EQ (0.001-0.1 µM) strongly increased the estrogenic response compared to the single substances.

Consumption of soy isoflavones during the prepubertal phase delays puberty and causes hypergonadotropic hypogonadism with disruption of hypothalamic-pituitary gonadotropins regulation in male rats.

Isoflavones are phytoestrogens with recognized estrogenic activity but may also affect testosterone, corticosterone and thyroid hormone levels in experimental models. However, the molecular mechanisms involved in these alterations are still unclear. Isoflavones are present in soy-based infant formula, in breast milk after the consumption of soy by the mother and are widely used for the preparation of beverages consumed by toddlers and teenagers. In this sense, we proposed to investigate the effects of soy isoflavone exposure during the prepubertal period, a recognized window of sensitivity for endocrine disruption, over the hypothalamic-pituitary-testicular (HPT) axis. For this, 42 3-week-old male Wistar rats were exposed to 0.5, 5 or 50 mg of soy isoflavones/kg from postnatal day (PND) 23 to PND60. We evaluated body growth, age at puberty, serum concentrations of LH, FSH, testosterone and estradiol, and the expression of the transcripts (mRNA) of genes encoding key genes controlling the hypothalamic-pituitary-testicular (HPT) axis. In the hypothalamus, we observed an increase in Esr1 mRNA expression (0.5 and 5 mg). In the pituitary, we observed an increase in Gnrhr mRNA expression (50 mg), a reduction in Lhb mRNA expression (0.5 mg), and a reduction in Ar mRNA expression. In the testis, we observed an increase in Lhcgr mRNA expression (50 mg) and a reduction in Star mRNA expression (0.5 and 5 mg). The serum levels of LH (5 and 50 mg) and FSH (0.5 mg) were increased, while testosterone and estradiol were reduced. Puberty was delayed in all groups. Taken together, these results suggest that prepubertal consumption of relevant levels of soy isoflavones disrupts the HPT axis, causing hypergonadotropic hypogonadism and altered expression levels of key genes regulating the axis.

Comprehending the Role of Endocrine Disruptors in Inducing Epigenetic Toxicity.

Endocrine disruptors are natural or man-made chemicals that interfere with the body's endocrine system leading to hormone synthesis and production defects. These chemicals are categorized as plasticizers and cosmetic chemicals, heavy metals, phytoestrogens, pesticides, detergents, surfactants, and flame retardants. Some of the most common endocrine disruptors are dioxins, bisphenol A, phthalates, perchlorate, perfluoroalkyl, and poly-fluoroalkyl substances (PFAs), phytoestrogens, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCB), triclosan, atrazine, lead, arsenic, mercury, organophosphate pesticides, and glycol ethers. Epigenetic alterations such as DNA methylation, histone modification, and miRNA regulation have been observed to play a major role in many diseases such as cancer, neurodegenerative diseases, PCOS, cardiovascular diseases, and various other disorders. In recent times, there has been a focus on endocrine-disrupting chemicals in epigenetic alterations. This review concentrates on estrogen and androgen disrupting effects, placental, and fetal effects, thyroid disrupting effects, and transgenerational effects of endocrine disruptors.

Elucidating the estrogen-like effects and biocompatibility of the herbal components in the Qing' E formula.

ETHNOPHARMACOLOGICAL RELEVANCE: The Qing' E Formula (QEF) is a compound preparation that was originally recorded in the 'Prescriptions of the Bureau of Taiping People's Welfare Pharmacy' during the Song Dynasty (10th century CE). It consists of four Chinese medicinal herbs, Eucommiae Cortex (Eucommia ulmoides), Psoraleae Fructus (Psoralea corylifolium), Juglandis Semen (Juglans regia), and Garlic Rhizoma. According to traditional Chinese medicine (TCM), QEF has the ability to tonify the kidney and strengthen muscle and bone. According to the 'kidney governing bone' theory in TCM, QEF is also used to treat the symptoms of climacteric syndrome, especially osteoporosis caused by reduced production of estrogen during the perimenopausal period; however, the therapeutic roles of the individual components of the QEF and their compatibility within the formula has not been investigated. AIM OF THE STUDY: In this study, the compatibility mechanism and estrogen-like action properties of the four herbal components in the QEF was elucidated according to the organizing principle of Chinese medicine formulas using both in vitro and in vivo models. MATERIALS AND METHODS: The estrogen-like effects of QEF and its herbal components were investigated in MCF7 and HEK293 cells as well as ovariectomized (OVX) rats. The estrogen-like effects of the QEF and its components were analyzed in vitro using Cell Counting Kit-8 and Luciferase reporter gene assays. In the in vivo studies, the blood plasma levels of hormones, lipids, neurotransmitters, aromatase, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured through enzyme-linked immunosorbent assays (ELISAs). The histological morphologies of the target organs after exposure to QEF were investigated by HE staining and immunohistochemical methods. The expression levels of estrogen pathway-related proteins and genes in the OVX rats were measured by Western blotting and real time quantitative PCR (RT-qPCR), respectively. RESULTS: The in vitro results showed that the QEF, Eucommia (EC) and Psoralea (PF) promoted the proliferation of MCF-7 cells and upregulated the expression of ERα, ERß and pS2 genes in the MCF-7 cells. Notably, the QEF demonstrated the most active estrogen-like effects compared to the individual ingredients. The in vivo results showed that the QEF, EC, and PF increased the uterine coefficient, upregulated the expression of both ERs (ERα and ERß) in the uterus, and increased blood serum hormone levels. QEF and its individual components ameliorated menopausal-derived lipid metabolism dysfunction, increased neurotransmitter production by stimulating the adrenal glands, enhanced the antioxidant activity in the serum by increasing the concentration of SOD, reversed ovariectomy-derived atrophy in the uterus, and reduced the weight gain associated with estrogen reduction in the OVX rats. The QEF also antagonize the loss of appetite of OVX animals caused by feeding Psoralea alone, which could explain the compatibility mechanism of Qing' E Formula with reducing toxicity and increasing efficiency. CONCLUSIONS: The estrogen-like effects of Eucommia and Psoralea were mainly mediated through activation of ERα and ERß. The phytoestrogen components regulated hormone production and the expression of related proteins and genes, which indicated that these components exhibited estrogen-like therapeutic effects. However, the QEF showed the greatest estrogen-like effects compared to the individual components. Overall, this corroborated the therapeutic prowess of the QEF and clarified the pharmacodynamic interactions between the different components extracts in the QEF.

Microbial Polymers as Sustainable Agents for Mitigating Health Risks of Plant-Based Endocrine Disruptors in Surface Water.

This study investigated the binding abilities of extracellular polymers produced by an environmentally isolated strain of Enterococcus hirae towards phytoestrogen endocrine disruptors-biochanin A, formonetin, genistein and daidzein. The extracellular biopolymer exhibited notable binding and removal for all four phytoestrogens, with a maximum removal of daidzein (87%) followed by genistein (72%) at a 1-1.5 mg/mL concentration. Adsorption proceeded rapidly at ambient temperature. The adsorption data fitted well with the Langmuir isotherm. Based on the adsorption energy, the biopolymer binding of phytoestrogens was inferred as daidzein > genistein > biochanin A > formononetin. Toxicity of the biopolymer (5-250 µg/mL) evaluated using RAW 264.7 cell lines indicated no significant (p < 0.05) changes in viability. In biopolymer-challenged Caenorhabditis elegans previously exposed to daidzein, complete protection to developmental toxicity, such as reduced egg-laying capacity, egg viability and progeny counts of the worm, was observed. The results of this study offer valuable insights into understanding the potential role of microbial extracellular biopolymers in binding and removal of phytoestrogens with sustainable technological implications in modulating the toxic effect of high levels of endocrine disruptors in the environment.

Role of Endocrine-Disrupting Chemicals in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: A Comprehensive Review.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disorder, affecting around 25% of the population worldwide. It is a complex disease spectrum, closely linked with other conditions such as obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome, which may increase liver-related mortality. In light of this, numerous efforts have been carried out in recent years in order to clarify its pathogenesis and create new prevention strategies. Currently, the essential role of environmental pollutants in NAFLD development is recognized. Particularly, endocrine-disrupting chemicals (EDCs) have a notable influence. EDCs can be classified as natural (phytoestrogens, genistein, and coumestrol) or synthetic, and the latter ones can be further subdivided into industrial (dioxins, polychlorinated biphenyls, and alkylphenols), agricultural (pesticides, insecticides, herbicides, and fungicides), residential (phthalates, polybrominated biphenyls, and bisphenol A), and pharmaceutical (parabens). Several experimental models have proposed a mechanism involving this group of substances with the disruption of hepatic metabolism, which promotes NAFLD. These include an imbalance between lipid influx/efflux in the liver, mitochondrial dysfunction, liver inflammation, and epigenetic reprogramming. It can be concluded that exposure to EDCs might play a crucial role in NAFLD initiation and evolution. However, further investigations supporting these effects in humans are required.

Estrogenic Effects of Extracts and Isolated Compounds from Belowground and Aerial Parts of Spartina anglica.

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.

In vitro estrogenic, cytotoxic, and genotoxic profiles of the xenoestrogens 8-prenylnaringenine, genistein and tartrazine.

Phytoestrogens have been widely praised for their health-promoting effects, whereas synthetic environmental estrogens are considered a toxicological risk to human health. The aim of this study was therefore to compare in vitro the estrogenic, cytotoxic, and genotoxic profiles of three common estrogen-like endocrine-disrupting chemicals: the phytoestrogens 8-prenylnaringenine (8-PN) and genistein and the synthetic xenoestrogen tartrazine. As assessed by a yeast bioreporter assay and estrogen-dependent proliferative response in human mammary gland adenocarcinoma cell line (MCF-7), 8-PN showed the highest estrogen-like activity of the three compounds, followed by tartrazine and genistein. After 24-h incubation on MCF-7 cells, all three compounds exhibited low cytotoxicity in the lactate dehydrogenase assay and no genotoxicity in the micronucleus assay. These results demonstrate that 8-PN, genistein and tartrazine possess variable estrogenic activity but display little cellular toxicity in short-term tests in vitro. No difference between phytoestrogens and a synthetic xenoestrogen could be established.

Genistein Induces Adipogenic and Autophagic Effects in Rainbow Trout (Oncorhynchus mykiss) Adipose Tissue: In Vitro and In Vivo Models.

Soybeans are one of the most used alternative dietary ingredients in aquafeeds. However, they contain phytoestrogens like genistein (GE), which can have an impact on fish metabolism and health. This study aimed to investigate the in vitro and in vivo effects of GE on lipid metabolism, apoptosis, and autophagy in rainbow trout (Oncorhynchus mykiss). Primary cultured preadipocytes were incubated with GE at different concentrations, 10 or 100 µM, and 1 µM 17ß-estradiol (E2). Furthermore, juveniles received an intraperitoneal injection of GE at 5 or 50 µg/g body weight, or E2 at 5 µg/g. In vitro, GE 100 µM increased lipid accumulation and reduced cell viability, apparently involving an autophagic process, indicated by the higher LC3-II protein levels, and higher lc3b and cathepsin d transcript levels achieved after GE 10 µM. In vivo, GE 50 µg/g upregulated the gene expression of fatty acid synthase (fas) and glyceraldehyde-3-phosphate dehydrogenase in adipose tissue, suggesting enhanced lipogenesis, whereas it increased hormone-sensitive lipase in liver, indicating a lipolytic response. Besides, autophagy-related genes increased in the tissues analyzed mainly after GE 50 µg/g treatment. Overall, these findings suggest that an elevated GE administration could lead to impaired adipocyte viability and lipid metabolism dysregulation in rainbow trout.

In utero exposure to low doses of genistein and di-(2-ethylhexyl) phthalate (DEHP) alters innate immune cells in neonatal and adult rat testes.

BACKGROUND: Although humans are exposed to mixtures of endocrine disruptor chemicals, few studies have examined their toxicity on male reproduction. We previously found that fetal exposure to a mixture of the phytoestrogen genistein (GEN) and the plasticizer di(2-ethylhexyl) phthalate (DEHP) altered gene expression in adult rat testes. OBJECTIVES: Our goal was to investigate the effects of fetal exposure to GEN-DEHP mixtures at two doses relevant to humans on testicular function and transcriptome in neonatal and adult rats. MATERIALS AND METHODS: Pregnant SD rats were gavaged with vehicle, GEN or DEHP, alone or mixed at 0.1 and 10 mg/kg/day, from gestation day 14 to birth. Fertility, steroid levels, and testis morphology were examined in neonatal and adult rats. Testicular transcriptomes were examined by gene array and functional pathway analyses. Cell-specific genes/proteins were determined by quantitative real-time PCR and immunohistochemistry. RESULTS: GEN-DEHP mixtures increased the rates of infertility and abnormal testes in adult rats. Gene array analysis identified more genes exclusively altered by the mixtures than individual compounds. Altered top canonical pathways included urogenital/reproductive developmental and inflammatory processes. GEN-DEHP mixtures increased innate immune cells and macrophages markers at both doses and ages, more strongly and consistently than DEHP or GEN alone. Genes exclusively increased by the mixture in adult testis related to innate immune cells and macrophages included Kitlg, Rps6ka3 (Rsk2), Nr3c1, Nqo1, Lif, Fyn, Ptprj (Dep-1), Gpr116, Pfn2, and Ptgr1. DISCUSSION AND CONCLUSION: These findings demonstrate that GEN-DEHP mixtures at doses relevant to human induce adverse testicular phenotypes, concurrent with age-dependent and non-monotonic changes in testicular transcriptomes. The involvement of innate immune cells such as macrophages suggests immediate and delayed inflammatory responses which may contribute to testicular dysfunction. Moreover, these effects are complex and likely involve multiple interactions between immune and non-immune testicular cell types that will entail further studies.

Applicability of the OECD 455 in-vitro assay for determination of hERa agonistic activity of isoflavonoids.

The Organisation for Economic Co-operation and Development (OECD)-validated transactivation assay using the human estrogen receptor alpha (hERα) Hela9903 cell line is used for activity evaluation of hERα agonists and antagonists. Due to many advantages, this assay is broadly used as an initial screening process. However, response significantly higher from that of 17-ß estradiol (E2) was observed with phytoestrogens for concentrations commonly above 1 µM in previous studies. The main aim of this study was thus to ascertain the applicability of OECD protocol 455 for evaluation of estrogenic activity of natural flavonoids, including known phytoestrogens. The estrogenic activities of aglycones as well as of O-methylated and glycosylated flavonoids were evaluated. Supra-maximal luciferase activity was seen for most of the flavonoids tested at concentrations even below 1 µM. hERα-mediated luciferase expression was confirmed with the competition assay specified in OECD protocol 455. However, at concentrations above 1 µM, non-specific interactions were also observed. Instead of EC50 values, which could not be determined for most of the isoflavonoids tested, the concentrations corresponding to 10% (PC10) and 50% (PC50) of the maximum activity of the positive control, E2, were used for quantitative determination of estrogenic activities. Appropriate evaluation of the data obtained with the current OECD protocol 455 validated assay represents a valuable tool for initial screening of natural flavonoids for estrogenic activity.

[Effects of equol on proliferation of colorectal cancer HCT-15 cell].

OBJECTIVE: To investigate the effect of racemic equol and equol enantiomers on the proliferation of colorectal cancer HCT-15 cell and the potential mechanism. METHODS: 3-( 4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide( MTT) assay was used to detect the effect of different concentrations of racemic equol and equol enantiomers( 0, 0. 5, 1, 5 and 10 µmol/L) on the proliferation of colorectal cancer HCT-15 cell. Western blot was used to detect the expression of estrogen receptor( ER)and nuclear factor 2 related factor 2( Nrf2). RESULTS: Racemic equol and( R) equol inhibited the proliferation of HCT-15 cell in a dose-dependent manner, whereas( S) equol had no effect on the proliferation of HCT-15 cell. Racemic equol increased the expression of ERß and Nrf2, while( R) equol increased the expression of Nrf2. CONCLUSION: Racemic equol can inhibit the proliferation of HCT-15 cell through estrogenic and antioxidative activity. R equol can inhibit the proliferation of HCT-15 cell through antioxidative activity, while( S) equol has no effect on the proliferation of HCT-15 cell.

The effects of dietary levels of genistein on ovarian follicle number and gene expression.

Genistein is a phytoestrogen found in soy. We previously found that adult exposure to dietary levels of genistein affected gestation time, parturition time, litter size, pup weight, and pup mortality in CD-1 mice. The present study investigated the effects of adult genistein exposure on follicle number and gene expression in the ovaries of CD-1 mice. We found that exposure to genistein had no effect on follicle number, but it did affect the expression of apoptotic regulatory genes (Bax, Bcl-2, Bid, and Dffa) in the ovary.

The effects of the botanical estrogen, isoliquiritigenin on delayed spatial alternation.

Age-related declines in cognitive function can impair working memory, reduce speed of processing, and alter attentional resources. In particular, menopausal women may show an acceleration in the rate of cognitive decline as well as an increased vulnerability to brain diseases as estrogens may play a neuroprotective and neurotrophic role in the brain. To treat menopausal symptoms, many women turn to botanical estrogens that are promoted as a safe and natural alternative to traditional hormone replacement therapy. However, the majority of these compounds have not been systematically evaluated for efficacy and safety. The current study investigated the efficacy of the commercially available botanical estrogenic compound isoliquiritigenin (ISL) to alter performance on an operant working memory task, delayed spatial alternation (DSA). ISL is a compound found in licorice root that has been shown to have a wide range of effects on different biological systems, including estrogenic properties. This botanical is currently being used in over the counter dietary supplements. Middle-aged (12-month old) Long-Evans female rats were ovariectomized and orally dosed with either 0 mg, 6 mg, 12 mg or 24 mg of ISL 60 min before testing on the DSA task. The DSA task required the rat to alternate its responses between two retractable levers in order to earn food rewards. Random delays of 0, 3, 6, 9 or 18 s were imposed between opportunities to press. ISL treatment failed to alter DSA performance. Previous work from our research group has found that estrogenic compounds, including 17ß-estradiol and the botanical estrogen genistein impair performance on the DSA task. The goal of our botanical estrogens research is to find compounds that offer some of the beneficial effects of estrogen supplementation, without the harmful effects. This work suggests that ISL may not carry the cognitive risks associated with most other estrogenic compounds tested to date.

Preconception exposure to dietary levels of genistein affects female reproductive outcomes.

Genistein is a phytoestrogen found in soy and soy-based products. Previously, we found that genistein adversely affected estradiol levels and follicle growth in vitro. Proper hormone production and follicle growth are key regulators of normal fertility. Therefore, we hypothesized that genistein adversely affects female fertility and pregnancy outcomes. To test this hypothesis, we dosed sexually mature female CD-1 mice (35days) with 0, 300, 500, or 1000ppm genistein for 30, 60, 150, and 240days. At the end of the dosing periods, we measured mating rate, pregnancy rate, fertility rate, gestation time, parturition time, pup mortality, litter size, average pup weight, and estradiol and progesterone levels. We found that chronic, preconception exposure to genistein affects gestation time, parturition time, litter size, pup weight, and pup mortality. Additionally, genistein exposure for 240days appears to have a protective effect on fertility rate, but does not affect hormone levels in vivo.

In vitro screening for estrogenic endocrine disrupting compounds using Mozambique tilapia and sea bass scales.

A wide range of estrogenic endocrine disruptors (EDCs) are accumulating in the environment and may disrupt the physiology of aquatic organisms. The effects of EDCs on fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We used a simple non-invasive assay to evaluate the impact of estrogens and EDCs on sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. These were exposed to estradiol (E2), two phytoestrogens and six anthropogenic estrogenic/anti-estrogenic EDCs and activities of enzymes related to mineralized tissue turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase) were measured. Semi-quantitative RT-PCR detected the expression of both membrane and nuclear estrogen receptors in the scales of both species, confirming scales as a target for E2 and EDCs through different mechanisms. Changes in TRAP or ALP activities after 30minute and 24h exposure were detected in sea bass and tilapia scales treated with E2 and three EDCs, although compound-, time- and dose-specific responses were observed for the two species. These results support again that the mineralized tissue turnover of fish is regulated by estrogens and reveals that the scales are a mineralized estrogen-responsive tissue that may be affected by some EDCs. The significance of these effects for whole animal physiology needs to be further explored. The in vitro fish scale bioassay is a promising non-invasive screening tool for E2 and EDCs effects, although the low sensitivity of TRAP/ALP quantification limits their utility and indicates that alternative endpoints are required.

Synthesis and characterization of genistein conjugated with gold nanoparticles and the study of their cytotoxic properties.

Gold nanoparticles conjugated with drug substances are used in diagnostics and therapies. Apart from the combinations involving gold nanoparticles conjugated with drug substances through linkers, a direct bonding is also known. In our paper the example of such a direct bonding between gold nanoparticles and genistein (AuNPs-GE) is presented. This conjugate was obtained in a one-pot synthesis and the formation of AuNPs-GE was monitored in terms of color change and UV-Vis spectroscopy. It has been shown that genistein reduces Au3+ ions to spherical Au0 nanocrystallites and acts as a stabilizing agent. The efficiency of the purification of the conjugate from free genistein was controlled by the capillary electrophoresis. Gold nanoparticles are homogeneously shaped and have a narrow range of size from 14 to 33nm and the size of the nanoparticles modified with genistein is around 64.64±0.41nm, as measured by the TEM and DSL techniques, respectively. The zeta potential of the gold nanoparticles modified with genistein is -19.32±0.82mV and suggests a high stability of the nanoparticles and lower toxicity for the normal cells. The identity of genistein on the gold nanoparticles was proved by the electrochemistry, NMR and Raman spectroscopy. The mechanism of the conjugate forming has been proposed. The coverage of gold nanoparticles with genistein 5.09% (m/m) has been calculated from the TGA analysis. Moreover, it has been proved that the obtained conjugate is characterized by a high cytotoxic activity towards cancer cells, as observed in the cell line test.

Activity based evaluation of a traditional Ayurvedic medicinal plant: Saraca asoca (Roxb.) de Wilde flowers as estrogenic agents using ovariectomized rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Saraca asoca (Roxb.) de Wilde, Ashok, is a popular traditional plant used for gynecological disorders. In India, the juice of Ashok flowers is traditionally consumed as a tonic by women in case of uterine disorders. But despite the use, its estrogenic potency is not yet evaluated and thus lacks the scientific recognition and acclaim. AIM OF THE STUDY: This study is designed to investigate the estrogenic potential of standardized ethanolic extract of Saraca asoca flowers (SAF) using ovariectomized (OVX) female albino Wistar rat model. MATERIALS AND METHODS: Saraca asoca flowers were extracted in ethanol using hot maceration technique and the extract was standardized in terms of content of four phytoestrogens like quercetin, kaempferol, ß-sitosterol and luteolin using HPTLC technique. Safety of the extract was evaluated at a dose of 2000mg/kg body weight in female albino Wistar rats as per the OECD guidelines. Bilateral ovariectomy surgery was performed for the excision of both the ovaries. The OVX animals were treated with the ethanolic extract of SAF at three dose levels- 100mg/kg, 200mg/kg and 400mg/kg body weight in distilled water as a vehicle, orally once a day for two weeks. Estradiol valerate was employed as a modern drug for comparative evaluation of the results. Estrogenic potency was studied by assaying the activities of serum and plasma marker enzymes and hormones viz. G6PDH, LDH, 17ß-estradiol, progesterone along with cholesterol, triglycerides and HDL, and vaginal cornification. The uterotrophic effect was evaluated by studying the histoarchitecture of the uterus, effect on uterine weight and changes in the levels of uterine glycogen content. RESULTS: HPTLC revealed the presence of markers like quercetin, kaempferol, ß-sitosterol and luteolin from the ethanolic extract of SAF. The content of the four markers was found to be 1.543mg/g, 0.924mg/g, 4.481mg/g and 2.349mg/g, respectively. SAF extract was found to be safe at an oral dose of 2000mg/kg body weight in rats. Among the three doses administered to ovariectomized rats, treatment with high dose was found to be more efficacious when compared with ovariectomized rats. CONCLUSION: The findings of this study firmly support the estrogenic potency of ethanolic extract of SAF which may be by the reason of phytoestrogens.