Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P < 0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P < 0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P < 0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P < 0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.     

Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes)

This study was designed to determine the estrogenic effect of the phytoestrogen genistein on several measures of endocrine function in adult Japanese medaka (Oryzias latipes) relative to 17-beta-estradiol. Adult animals of both sexes were exposed to 75, 750 and 30,000 ng/fish (average fish weight equals 0.26 g) of genistein by i.p. injection, with a positive control group treated with 300 ng/fish of 17-beta-estradiol, while a negative control group received a vehicle-only (corn oil) injection. Content of vitellogenin, the yolk glycoprotein made in the liver in response to estradiol stimulation, was measured using Western blots. Circulating estradiol and testosterone levels were measured using a steroid-enzyme immunosorbant assay. The ability of ovaries and testes to synthesize and release estradiol and testosterone was determined by ex vivo incubation of gonads with 25-hydroxycholesterol. Vitellogenin, while induced by 17-beta-estradiol, was not increased in the liver of individuals treated with genistein. In females, genistein treatment at 750 and 30,000 ng increased the estradiol production of ovaries more than the 17-beta-estradiol treatment. In males, genistein treatment resulted in decreased testosterone production from ex vivo testis and a comparable reduction in circulating testosterone level. The changes in vitellogenin, circulating steroids and ex vivo steroidogenesis in medaka in response to genistein are similar to that of 17-beta-estradiol. However, some endpoints are more sensitive to estradiol treatment (vitellogenin), while others are more sensitive to genistein (male testosterone and ovarian estrogenesis).     

The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri

Various phytoestrogens such as formononetin, daidzein, genistein and equol were synthesized. Their purity was assessed by various analytical techniques including melting point determination, thin-layer chromatography (TLC), infra-red spectra (i.r. spectra), nuclear magnetic resonance (1H- and 13C-NMR) and gas chromatography coupled with mass spectrometry (GC-MS). The estrogenic activity of these compounds, as well as biochanin A and coumestrol, was biologically tested by the induction of vitellogenin secretion in yearling sturgeon and compared to the activity of estradiol-17 beta. Pure daidzein, biochanin A, genistein, equol and coumestrol all had estrogenic activity as assessed by their induction of hepatic synthesis of vitellogenin when administrated intraperitoneally to yearling Siberian sturgeon. Coumestrol seemed to be the most potent compound, inducing the most vitellogenin secretion with the lowest dose administered. Formononetin was inactive when administered by the intraperitoneal route. All the phytoestrogens tested were considerably less potent than estradiol-17 beta.     

Toxicity to early life stages and an estrogenic effect of a bisphenol A metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on the medaka (Oryzias latipes)

In a recent study, it was reported that 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a metabolite of bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane), showed estrogenic activity in several in vitro assays, and the estrogenic activity of MBP was higher than that of BPA. In this study, we have investigated the early life stage toxicity and estrogenic effect of MBP on medaka (Oryzias latipes). The 96-h median lethal concentration value of MBP and BPA with 24-h-old larvae was estimated to be 1,640 and 13,900 microg/l, respectively. The hatchability of fertilized eggs exposed to MBP and BPA over 14 days was significantly decreased at doses of 2,500 microg/l and 12,500 microg/l, respectively. Moreover, to compare the potency of estrogenic activity in vivo, male medaka were exposed to various concentrations of MBP and BPA for 21 days. The lowest-observed-effect concentrations of MBP and BPA for hepatic vitellogenin induction in male medaka were estimated to be 4.1 and 1,000 microg/l, respectively. These results suggest that MBP has high toxicity for early life stages of the medaka, and that the estrogenic activity of MBP was about 250-fold higher than that of BPA to male medaka.     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Selecting the appropriate rodent diet for endocrine disruptor research and testing studies

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.     

Survey of estrogenic activity in fish feed by yeast estrogen-screen assay

Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.     

Novel Approach for Evaluation of Estrogenic and Anti‐Estrogenic Activities of Genistein and Daidzein using B16 Melanoma Cells and Dendricity Assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti‐estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17β‐estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est >> genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50 000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti‐estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti‐estrogenic activity in vitro.     

Dietary daidzein,but not genistein,has a hypocholesterolemic effect in non-ovariectomized and ovariectomized female Sprague-Dawley rats on a cholesterol-free diet

We compared the effects of two major isoflavones, daidzein and genistein, on lipid metabolism in rats. Daidzein (150 mg/kg diet), genistein (150 mg/kg diet), daidzein and genistein (1:1, 300 mg/kg diet), or control diets were fed to 4 groups of 6-week-old ovariectomized (Ovx) and non-Ovx Sprague Dawley rats for 4 weeks. Dietary daidzein, but not genistein, reduced serum and hepatic total cholesterol levels significantly relative to that by the control group, regardless of whether the rats had undergone ovariectomy. Genistein did not exhibit any physiological effects on lipid levels, but did affect genes involved in cholesterol metabolism. These results indicate that daidzein and genistein may influence lipid regulation via differing modes of action.     

Growth performance, carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782 microg/g in SBM and 125 microg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4 +/- 1.1 kg were allotted to flat deck boxes. During the growing/finishing period (days 70-170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239 +/- 44; daidzein 162 +/- 42; equol 12 +/- 4 ng/ml plasma) and animals fed SPC (genistein 22 +/- 9 and daidzein 8 +/- 3, and equol 10 +/- 3 ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.     

Novel approach for evaluation of estrogenic and anti-estrogenic activities of genistein and daidzein using B16 melanoma cells and dendricity assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti-estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17 beta-estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est > genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50,000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti-estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti-estrogenic activity in vitro.     

Endocrine disrupting activity in fruits and vegetables evaluated with the E-screen assay in relation to pesticide residues

Food is likely to be one of the most important routes of human exposure to endocrine disrupting compounds (EDCs). In the present study, we evaluated the total estrogenic activity of fruits and vegetables, which was calculated using the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen), in relation to pesticide residues. We analysed 44 food samples, 30 fruits and 14 vegetables. Of these samples, 10 did not contain any pesticide residues. The other 34 samples contained from 1 to 7 pesticide residues in concentrations ranging from 0.03 to 1.91 ppm. Estrogenic activity was detected in the 59% of samples tested. The positive controls used were 17-β-estradiol (E2), the phytoestrogen genistein and the pesticide endosulfan. The average value of estradiol equivalency quantity (EEQ) for all positive samples was 0.15±0.32 μg/100g. A low correlation was found between the concentration of pesticide residues and the EEQ values (Spearman correlation r=0.376 and p=0.012). Using values obtained from the literature, we compared the estrogenic activity of food samples with the intrinsic content of phytoestrogens, but we found no correlations. Our results also suggested that the calculated intake of dietary EDCs might represent a concentration comparable to the normal endogenous estrogen concentration in human blood.     

Genistein and daidzein modulate in vitro rat uterine contractile activity

The present study investigated the effect of genistein, daidzein and estradiol on in vitro rat uterine responsiveness to oxytocin (OT) and PGF(2)alpha or luprostiol (L). In a first experiment, animals were either sham-operated (SH; n=5), or ovariectomized (OVX; n=20) and orally treated for three months with either genistein (G; n=5; 10 microg/g BW/d) or daidzein (D; n=5; 10 microg/g BW/d) or 17 alpha-ethinylestradiol (E; n=5; 23 microg/kg BW/d) or untreated (OVX; n=5). At necropsy, the basal uterine tension was lower in OVX, G and D than in SH, the highest value being measured in E. Oxytocin (10(-12); 10(-11) M) or PGF(2)alpha (10(-12); 10(-9) M) induced an increase in SH, but not in OVX, E and G. In D, only the highest doses were efficient. In a second experiment, 20 intact animals were s.c. injected with either genistein (G; n=5; 10 microg/g BW) or daidzein (D; n=5; 10 microg/g BW) or estradiol benzoate (E; n=5; 23 microg/kg BW) or vehicle (C: controls; n=5), and killed 24 h later. In C and E, OT (10(-15) to 10(-10) M) or L (10(-12) to 10(-7) M) stimulated uterine contractile activity in a dose-dependent manner until a maximal level. On the opposite, in G and D, contractile agents (except the highest luprostiol doses) did not stimulate myometrium contractions. Moreover, radioligand binding assays showed that genistein or daidzein inhibited the specific binding of [(3)H] estradiol to the calf uterus estrogen receptor (ER). Therefore, it could be postulated that both genistein and daidzein might bind to the rat uterus ER, inducing either anti-estrogenic or very weak estrogenic effects (depending on the experimental conditions) on in vitro uterine responsiveness to OT and PGF(2)alpha or luprostiol.     

Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test

Six isoflavones, daidzein (4',7,-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), genistin (genistein 7-O-beta-D-glucopyranoside), isoprunetin (4',7-dihydroxy, 5-metoxyisoflavone), isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside and four flavones, luteolin (3',4',5,7-tetrahydroxyflavone), luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C (4',5,7-trihydroxy,8-isoprenylflavone) were purified from Mediterranean plants (Genista morisii and Genista ephedroides) and their estrogenic activity was assessed by a yeast reporter gene assay (Saccharomyces cerevisiae RMY326 ER-ERE). Licoflavone C showed a powerful estrogenic activity at 10(-7) M (0.0338 microg/ml) and it was 47.45% than 10(-8) M 17beta-estradiol (0.00272 microg/ml). The estrogenicity of this flavone was found to be comparable to the activity showed by genistein at 10(-6) M (0.27 microg/ml). This study points out that a glucose substituent in flavones and isoflavones modulates the hormone-like activity in a different way. Isoflavone aglycones showed a more estrogenic activity than the corresponding glucosides. Conversely, the glucosidation made estrogenic the flavone luteolin and the position of substitution differently influenced the estrogenic activity of compounds.     

Genistein and daidzein modulate hepatic glucose and lipid regulating enzyme activities in C57BL/KsJ-db/db mice

This study examines whether anti-diabetic effects of genistein and daidzein are mediated by hepatic glucose and lipid regulating enzyme activities in type 2 diabetic animals. Male C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and age-matched non-diabetic littermates (db/+) were used in this study. The db/db mice were divided into control, genistein (0.02%, w/w) and daidzein (0.02%, w/w) groups. The blood glucose and HbA(1c) levels were significantly lower in the genistein and daidzein groups than in the control group, while glucose tolerance only was significantly improved in the genistein-supplemented group. The plasma insulin and C-peptide levels did not differ significantly between groups, yet the glucagon level was lower in the genistein and daidzein groups compared to that in the control db/db or db/+ group. The genistein and daidzein supplements increased the insulin/glucagon ratio in the type 2 diabetic animals. While the hepatic glucokinase activity was significantly lower in the db/db control group, the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were significantly higher in the control group compared to the db/+ group. Interestingly, these hepatic glucose metabolizing enzyme activities were reversed by the genistein and daidzein supplementation in db/db mice compared to the control group. The hepatic fatty acid synthase, beta-oxidation and carnitine palmitoyltransferase activities were all significantly lower in the genistein and daidzein groups than in the control group. The genistein and daidzein supplements also improved the plasma total cholesterol, triglyceride, HDL-cholesterol/total cholesterol, free fatty acid and hepatic triglyceride concentrations in the db/db mice. These results suggest that genistein and daidzein exert anti-diabetic effect in type 2 diabetic conditions by enhancing the glucose and lipid metabolism.