In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.     

Potential tissue selectivity of dietary phytoestrogens and estrogens

The recent discovery of a second estrogen receptor subtype, estrogen receptor-beta, may significantly advance our understanding of tissue specific effects of estrogenic compounds, both natural and synthetic. Although specific effects mediated by estrogen receptor beta in vivo remain to be elucidated, hypothetically the existence of two estrogen receptor subtypes (differing in both tissue distribution and biological activity) may help to explain the curious pharmacological behaviour of many estrogenic and antiestrogenic compounds, including the naturally occurring dietary phytoestrogens.     

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.     

Estrogenic plant foods of red colobus monkeys and mountain gorillas in Uganda

Phytoestrogens, or naturally occurring estrogen-mimicking compounds, are found in many human plant foods, such as soybeans (Glycine max) and other legumes. Because the consumption of phytoestrogens may result in both health benefits of protecting against estrogen-dependent cancers and reproductive costs of disrupting the developing endocrine system, considerable biomedical research has been focused on the physiological and behavioral effects of these compounds. Despite this interest, little is known about the occurrence of phytoestrogens in the diets of wild primates, nor their likely evolutionary importance. We investigated the prevalence of estrogenic plant foods in the diets of two folivorous primate species, the red colobus monkey (Procolobus rufomitratus) of Kibale National Park and mountain gorilla (Gorilla beringei) of Bwindi Impenetrable National Park, both in Uganda. To examine plant foods for estrogenic activity, we screened 44 plant items (species and part) comprising 78.4% of the diet of red colobus monkeys and 53 plant items comprising 85.2% of the diet of mountain gorillas using transient transfection assays. At least 10.6% of the red colobus diet and 8.8% of the gorilla diet had estrogenic activity. This was mainly the result of the red colobus eating three estrogenic staple foods and the gorillas eating one estrogenic staple food. All estrogenic plants exhibited estrogen receptor (ER) subtype selectivity, as their phytoestrogens activated ERβ, but not ERα. These results demonstrate that estrogenic plant foods are routinely consumed by two folivorous primate species. Phytoestrogens in the wild plant foods of these two species and many other wild primates may have important implications for understanding primate reproductive ecology.     

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.     

Prenylation has a compound specific effect on the estrogenicity of naringenin and genistein

A variety of plant derived substances, so-called phytoestrogens (PEs), although structurally not related to steroids, produce effects similar to the mammalian estradiol. However, little is known so far about the structural requirements which determine PE activities. Taking into consideration that prenylation reactions are relatively common in plant secondary metabolism, the activity of a set of three PE derivatives of genistein and naringenin, namely genistein, 8-prenylgenistein (8PG), 6-(1,1-dimethylallyl)genistein (6DMAG), naringenin, 8-prenylnaringenin (8PN) and 6-(1,1-dimethylallyl)naringenin (6DMAN) was compared regarding structure–estrogenicity relationships in three functionally different estrogen receptor assays.Strong estrogenic activities were recorded for 6DMAN and 8PN in all assays used, while the parent compound naringenin showed only very weak estrogenicity.In contrast, in the case of genistein derivatives, only genistein itself exhibited estrogenic activity in a yeast based assay. In MVLN breast cancer cells, a bioluminescent MCF-7-derived cell line, the estrogenic activity of all three genistein derivatives was similar. Studying alkaline phosphatase activity in Ishikawa endometrial cancer cells as an estrogenic response marker revealed a similar pattern of estrogenicity of the genistein derivatives compared to the yeast based assay although a slight estrogenic effect of 6DMAG and 8PG was apparent.In summary, this study demonstrates that prenylation often found in plant secondary metabolism differentially modifies estrogenic properties of PEs depending on the basic structure of the respective PE.     

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.     

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.     

Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens

Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner. For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used. The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model. We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p′-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa. We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line. Estradiol, which induced AlkP at levels as low as 10⁻⁸ M, was used as positive control. Most of the compounds studied showed a clear dose-dependent estrogenic effect. Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2–4-fold at a concentration of 10⁻⁶ M. The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10⁻⁶ M, octylphenol at 10⁻⁵ M. Effects of o,p′-DTT could not be measured. ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects. The latter result demonstrated the estrogen receptor dependency of this process. In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.     

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.     

Additive mixture effects of estrogenic chemicals in human cell-based assays can be influenced by inclusion of chemicals with differing effect profiles

A growing body of experimental evidence indicates that the in vitro effects of mixtures of estrogenic chemicals can be well predicted from the estrogenicity of their components by the concentration addition (CA) concept. However, some studies have observed small deviations from CA. Factors affecting the presence or observation of deviations could include: the type of chemical tested; number of mixture components; mixture design; and assay choice. We designed mixture experiments that address these factors, using mixtures with high numbers of components, chemicals from diverse chemical groups, assays with different in vitro endpoints and different mixture designs and ratios. Firstly, the effects of mixtures composed of up to 17 estrogenic chemicals were examined using estrogenicity assays with reporter-gene (ERLUX) and cell proliferation (ESCREEN) endpoints. Two mixture designs were used: 1) a 'balanced' design with components present in proportion to a common effect concentration (e.g. an EC(10)) and 2) a 'non-balanced' design with components in proportion to potential human tissue concentrations. Secondly, the individual and simultaneous ability of 16 potential modulator chemicals (each with minimal estrogenicity) to influence the assay outcome produced by a reference mixture of estrogenic chemicals was examined. Test chemicals included plasticizers, phthalates, metals, PCBs, phytoestrogens, PAHs, heterocyclic amines, antioxidants, UV filters, musks, PBDEs and parabens. In all the scenarios tested, the CA concept provided a good prediction of mixture effects. Modulation studies revealed that chemicals possessing minimal estrogenicity themselves could reduce (negatively modulate) the effect of a mixture of estrogenic chemicals. Whether the type of modulation we observed occurs in practice most likely depends on the chemical concentrations involved, and better information is required on likely human tissue concentrations of estrogens and of potential modulators. Successful prediction of the effects of diverse chemical combinations might be more likely if chemical profiling included consideration of effect modulation.     

The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties

As the population ages, there is an ever-increasing need for therapeutic agents that can be used safely and efficaciously to manage symptoms related to postmenopausal estrogen deficiency. Endogenous estrogens, e.g., 17beta-estradiol, of exogenous mammalian origin, e.g., horses, have long been used to manage such symptoms. There are more than 20 different classes of phytochemicals that have demonstrated affinity for human estrogen receptors in vitro. Some studies on exogenous estrogenic substances of botanical origin (phytoestrogens), such as standardized formulations of plant extracts with in vitro and in vivo estrogenic activity from soy (Glycine max Merill.) and red clover (Trifolium pratense L.), suggest clinical efficacy. Few clinical data for phytoestrogens other than isoflavonoids are available. In an exhaustive review of the literature through 2003, only two clinical trials were identified that were designed to evaluate the effect of hops (Humulus lupulus L.) on symptoms related to menopause. Folkloric, chemical, and biological literature relating primarily to the use of hops for their estrogenic activity, and two human clinical trials, are reviewed.     

Design, synthesis, and evaluation of peptides with estrogen-like activity

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC(50) 1 microM) and estrogen receptor (ER)alpha (IC(50) 500 microM) but not ERbeta, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC(50) of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC(50) of 100 microM for ERalpha, and 100-250 microM for ERbeta), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands.     

Novel D-ring modified steroid derivatives as potent,non-estrogenic,steroid sulfatase inhibitors with in vivo activity

In pursuit of novel steroid sulfatase (STS) inhibitors devoid of estrogenicity, several D-ring modified steroid derivatives were synthesised. In vitro evaluation of the compounds identified two highly potent inhibitors, 4a and 4b, which were 18 times more active than estrone-3-O-sulfamate (EMATE), both having IC(50) values of ca. 1nM. These 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives were synthesised from estrone, via the intermediate 1, which was easily alkylated, deprotected and sulfamoylated affording the final compounds in high yields. In order to assess their biological profile, the selected inhibitors were tested for their in vivo inhibitory potency and estrogenicity in ovariectomised rats. After an oral dose of 10mg/kg per day for 5 days, 4a and 4b were found to inhibit rat liver steroid sulfatase by 99%. They were also devoid of estrogenic activity in the uterine weight gain assay, indicating that these two leads have therapeutic potential for the treatment of hormone-dependent breast cancer.     

Naringenin-type flavonoids show different estrogenic effects in mammalian and teleost test systems

The estrogenic activity of several intermediary plant compounds has raised concern about possible risks of unwanted interference with endocrine regulation, but on the other hand there are potential medical benefits, in particular in treatment of menopausal symptoms or cancer. In the present study, we compare the estrogenic effects of phytoestrogens naringenin, 8-prenylnaringenin, 6-(1,1-dimethylallyl)naringenin, and the synthetic 4'-acetyl-7-prenyloxynaringenin. Two mammalian in vitro systems and a fish in vivo system were used to study the estrogenic properties with reference to genistein, 17-beta-estradiol or ethynylestradiol. Strong differences were observed between the mammalian in vitro and the fish in vivo test system. In the medaka sex reversal/vtg gene expression assay no estrogenic effects of the naringenin-type flavonoids were observed, while mammalian in vitro systems showed a similar and graded response to the test compounds.