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.     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.     

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.     

反相高效液相层析法测定新鲜植物材料中的植物雌激素含量

通过适当的样品处理方法,游离的和结合的植物雌激素［大豆素,雌马酚,染料木素,芒柄花素,香豆雌酚和美皂异黄酮］被从新鲜植物材料的提取物中分离出来,并在不同的紫外光波长下,可被ＨＰＬＣ法定量测定,根据滞留时间和标准品的添加,而鉴别出植物雌激素的层析波峰。本方法的测定灵敏度为２ｐｐｍ。白三叶草样品的加样回收率在８０％－１００％之间（平均回收率变异系数为５．４％）。通过比较游离植物雌激素的含量测定,本方法     

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.     

Phytoestrogens directly inhibit TNF-α-induced bone resorption in RAW264.7 cells by suppressing c-fos-induced NFATc1 expression

TNF-α-induced osteoclastogenesis is central to post-menopausal and inflammatory bone loss, however, the effect of phytoestrogens on TNF-α-induced bone resorption has not been studied. The phytoestrogens genistein, daidzein, and coumestrol directly suppressed TNF-α-induced osteoclastogenesis and bone resorption. TRAP positive osteoclast formation and resorption area were significantly reduced by genistein (10(-7) M), daidzein (10(-5) M), and coumestrol (10(-7) M), which was prevented by the estrogen antagonist ICI 182,780. TRAP expression in mature TNF-α-induced osteoclasts was also significantly reduced by these phytoestrogen concentrations. In addition, in the presence of ICI 182,780 genistein and coumestrol (10(-5) -10(-6) M) augmented TNF-α-induced osteoclast formation and resorption. However, this effect was not observed in the absence of estrogen antagonist indicating that genistein's and coumestrol's ER-dependent anti-osteoclastic action normally negates this pro-osteoclastic effect. To determine the mechanism mediating the anti-osteoclastic action we examined the effect of genistein, coumestrol, and daidzein on caspase 3/7 activity, cell viability and expression of key genes regulating osteoclast differentiation and fusion. While anti-osteoclastic phytoestrogen concentrations had no effect on caspase 3/7 activity or cell viability they did significantly reduce TNF-α-induced c-fos and NFATc1 expression in an ER dependent manner and also inhibited NFATc1 nuclear translocation. Significant decreases in NFκB and DC-STAMP levels were also noted. Interestingly, constitutive c-fos expression prevented the anti-osteoclastic action of phytoestrogens on differentiation, resorption and NFATc1. This suggests that phytoestrogens suppress TNF-α-induced osteoclastogenesis via inhibition of c-fos-dependent NFATc1 expression. Our data provides further evidence that phytoestrogens have a potential role in the treatment of post-menopausal and inflammatory bone loss directly inhibiting TNF-α-induced resorption.     

湖南八大公山的植物区系及其在植物地理学上的意义

湖南八大公山自然保护区是华中地区面积大且植物保存较完好的地点。土著种子植物计162科、709属、1775种。全部属按15个地理分布型进行分析,全部种按更细的分布型类别（中国特有分为10个亚型）进行分析。结果表明,本地热带分布的科属虽为数不少,但温带分布的科属更显优势,北温带科属比较集中,同时中国、东亚、及东亚一北美特有属也很集中。本山中国特有种计1120种,华中特有种为347种。植物区系的古老、残遗及特有性突出,可视为中国（华中）有典型代表性的山地。     

Effects of a normal, human-concentration, phytoestrogen diet on rat uterine growth.

The estrogenic action of the prototype natural phytoestrogen coumestrol was examined in rats in in vitro and in vivo tests. To establish the binding specificity of coumestrol and its relation to biological activities, saturation analyses and uterine weight assays were performed. These assays indicated that coumestrol competitively inhibited binding to the estrogen receptor and induced increases in uterine weight in keeping with its estrogen receptor affinity constant. Most importantly, coumestrol was uterotrophic when incorporated in a semipurified diet at natural dietary concentrations. Significant increases occurred in both uterine wet weight and dry weight, indicating that coumestrol produces true uterine growth. Effects appeared to be cumulative, raising questions of time-related interactions with other estrogen-sensitive mechanisms and clearance of isoflavonoids. Coumestrol induced uterine growth over a 90-hour period at dietary concentrations of 0.01 to 0.1%. Lower doses not active over this period were active when provided over a longer period of time: a 0.005% concentration was not active over a 90-hour period, but was active when provided over a 180-hour period. Coumestrol-induced uterine growth was accompanied by the induction of cytosolic progestin receptors and increases in nuclear estrogen binding. Scatchard analyses verified that these changes were due to changes in receptor number. These studies show that the naturally occurring phytoestrogens have dramatic estrogenic effects at natural dietary levels. These actions may be expressed via traditional receptor-mediated actions and therefore may have the same implications for development, health, and disease as do the steroidal estrogens produced by the body. Because rats have no sex hormone-binding globulin, further studies must be conducted in humans. However, these findings suggest that the natural dietary phytoestrogen coumestrol is a potent estrogen that must be considered in calculating the total estrogenic load to which humans are exposed during normal life.     

Acute effects of three isoflavone class phytoestrogens and a mycoestrogen on cerebral microcirculation

Phytoestrogens and mycoestrogens are naturally occurring plant and fungus secondary metabolites with estrogen-like structure and/or actions. We aimed to check the hypothesis that phytoestrogens and mycoestrogens, due to their ability to elicit cerebral vasodilation, can induce acute increases in brain blood perfusion. For this purpose, we continuously recorded cerebrocortical perfusion by laser-Doppler flowmetry in anesthetized rats receiving intracarotid infusions (1 mg/kg) of one of the following estrogenic compounds: biochanin A, daidzein, genistein or zearalanone. We have shown the ability of two isoflavone class phytoestrogens (daidzein and biochanin A) and the mycoestrogen zearalanone to induce acute increases in brain blood flow when locally infused into the cerebral circulation of anesthetized rats. The isoflavone genistein failed to induce a significant increase in brain perfusion. No concomitant changes in blood pressure were recorded during the cerebral effects of the estrogenic compounds. Therefore, these microcirculatory effects were due to direct actions of the estrogenic compounds on the cerebrovascular bed.     

Interaction of phytoestrogens and other environmental estrogens with prostaglandin synthase in vitro

The phytoestrogens daidzein, genistein, equol and coumestrol were found to stimulate microsomal prostaglandin H synthase (PHS) in vitro in a concentration-dependent manner when PHS-activity was measured by arachidonic acid-dependent oxygen uptake. These compounds were co-oxidized by PHS and the conversion of parent compounds was measured by HPLC analysis. The stimulation of PHS-cyclooxygenase by these compounds was partially reversed at high concentrations probably due to their antioxidant properties causing inhibition. In contrast, the monomethyl ethers of daidzein and genistein, formononetin and biochanin A, had little or weakly inhibitory effect on PHS, and appear to be no or poor co-substrates for PHS. Compared to the equine estrogen equilin, its metabolite d-equilenin was poorly metabolized by PHS and inhibited rather than stimulated PHS-cyclooxygenase activity in vitro. The resorcylic acid lactones zearalenone and zeranol, on the other hand, were surprisingly good inhibitors of PHS-cyclooxygenase. Furthermore, zeranol inhibited both the arachidonic acid and the hydrogen-peroxide-dependent oxidation of DES in contrast to indomethacin which inhibited only cyclooxygenase-dependent co-oxidation of DES. The results of this in vitro study are discussed in the context of data on synthetic and steroidal estrogens and support the idea that PHS-activity may be modulated by interaction with certain estrogenic compounds.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

Behavioral effects of endocrine-disrupting substances: phytoestrogens

A major source of endocrine-disrupting substances, usually not considered in laboratory animal experiments, is the diet used in research investigations. Soy represents the main protein source in almost all natural-ingredient commercially available formulated diets. Soy-derived isoflavones are the most abundant and in many ways the most studied phytoestrogens, and phytoestrogens (isoflavones) are known endocrine disruptors. Research is reviewed that identifies the physiological and behavioral endocrine-disrupting effects of dietary phytoestrogens (isoflavones) in animal diets, including most of the isoflavones, which are in a glycoside form and biologically inactive, and those in the gastrointestinal tract, which are biologically active. The isoflavones genistein and daidzein have similar molecular weights and structural characteristics to that of 17-beta estradiol, which may enable them to exert estrogenic and antiestrogenic properties are described and characterized. Daidzein can be further metabolized to the potent and abundant molecule equol, which in rodents is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones. Equol has the unique and important ability to specifically bind 5 alpha-dihydro-testosterone, and to act in turn to inhibit the action of this potent androgen. The specific influence of dietary soy phytoestrogens on consumptive, learning and memory, and anxiety-related behaviors is identified. Regulatory behaviors such as food and water intake, adipose deposition and leptin, and insulin levels affected by dietary isoflavones are also discussed.     

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

The effects of ACTH, phytoestrogens and estrogens on corticosterone secretion by gander adrenocortical cells in breeding and nonbreeding seasons

The aim of this study was to investigate the effects of ACTH, phytoestrogens (genistein, daidzein, biochanin A and coumestrol), and animal estrogens (estradiol and estrone) on corticosterone secretion by isolated adrenocortical cells of the ganders in breeding (April) and nonbreeding seasons (July). ACTH stimulated corticosterone output in the breeding season. In July (photorefractoriness and postbreeding molt) ACTH had no effect on corticosterone production. Coumestrol reduced corticosterone secretion by the cells obtained in nonbreeding season. Other examined phytoestrogens did not affect corticosterone production. Estrogens showed differentiated effects. Estradiol stimulated the corticosterone output in breeding season; estrone inhibited corticosterone release in July. The season can probably affect sensitivity of isolated gander adrenal cells, especially to ACTH. It seems that goose adrenocortical cells, in contrast to the mammalian cells, can be weakly sensitive to phytoestrogens.     

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.     

Daidzein and genistein content of fruits and nuts

Dietary phytoestrogens such as the isoflavones daidzein and genistein are thought to protect against chronic diseases that are common in Western societies, such as cancer, osteoporosis, and ischemic heart disease. In addition, there are concerns regarding the deleterious effects of hormone-like compounds, especially with respect to the development of infants. However, there is little information regarding the phytoestrogen content of foods, and therefore epidemiologic investigations of phytoestrogens are limited. As part of a study quantifying the consumption of phytoestrogens, the objective of this work was to assess the daidzein and genistein content of fruits and nuts commonly eaten in Europe. Eighty different fruits and nuts were sampled, prepared for eating, and freeze-dried. Daidzein and genistein were extracted from the dried foods, and the two isoflavones were quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 80 foods assayed, 43 contained no detectable daidzein or genistein, at a limit of quantification of 1 microg/kg dry weight of food. Nine foods contained more than 100 microg of the two isoflavones combined per kilogram wet weight, and 28 contained less than this amount. Currants and raisins were the richest sources of the isoflavones, containing 2,250 microg and 1,840 microg of the two isoflavones combined per kilogram of wet weight of food. Although fruits and nuts are not as rich in isoflavone phytoestrogens as are soy and other legumes, this is the first documentation of levels of daidzein and genistein occurring in these foods.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.