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.     

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.     

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.     

Impact of estradiol structural modifications (18-methyl and/or 17-hydroxy inversion of configuration) on the in vitro and in vivo estrogenic activity

It is well recognized that the majority of breast cancers are initially hormone-dependent and that 17β-estradiol (17β-E2) plays a crucial role in their development and progression. For this reason, using a compound able to block a specific enzyme involved in the last steps of the biosynthesis of 17β-E2 remains a rational way to treat estrogen-dependent diseases such as breast cancer. The present study describes the biological in vitro and in vivo evaluation of a structural modification (inversion of C18-methyl group at position 13 from β to α face) of 17β-E2 (1) and 17α-estradiol (17α-E2; 2). The two epimers 18-epi-17β-E2 (3) and 18-epi-17α-E2 (4) were obtained in two chemical steps by inversion of the C18-methyl of estrone using 1,2-phenylendiamine in refluxing acetic acid and reduction of ketone at position C17 with LiAlH(4). The new E2 isomers were tested on estrogen-sensitive cell lines (MCF-7 and T-47D), on estrogen-sensitive tissues (uterus and vagina of mice) and on estrogen receptor (ER) to determine their estrogenic potency relatively to natural estrogen 17β-E2 (1). The results show that 18-epi-17β-E2 (3) possesses the lower affinity for ER (RBA = 1.2%), the lower estrogenicity on estrogen-sensitive cells (1000 folds less estrogenic than 17β-E2 in MCF-7) and no uterotrophic (estrogenic) activity when tested on mice. In fact, we observed the following order of estrogenicity: 18-epi-17β-E2 (3)<18-epi-17α-E2 (4) < 17α-E2 (2)17β-E2 (1). These results suggest that the inversion of C18-methyl of natural 17β-E2 scaffold could be a useful strategy to decrease the estrogenicity of E2 derivatives used as enzyme inhibitors in the context of a treatment of estrogen-dependent diseases.     

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.     

高通量筛选雌激素类化合物重组绿色荧光蛋白酵母细胞测评体系

以载体双表达的方式构建重组酵母环境雌激素的评价体系, 用于快速筛选雌激素类化合物。在表达载体中, 用3-磷酸甘油醛脱氢酶(GPD)启动子驱动a人雌激素受体基因(hERa)的表达; 在报告载体中, 用雌激素效应元件(ERE)调控的绿色荧光蛋白(yEGFP)作为报告基因。将两者转化于酵母细胞(W303-1A)中, 构建成重组绿色荧光蛋白酵母细胞。该酵母细胞经不同浓度的雌激素类化合物作用后, 发现GFP的表达量与此类受试物具有明显的剂量效应关系。与其他环境雌激素酵母评价体系相比, 该重组酵母评价细胞, 在应用时不需要破坏细胞壁, 也不需要底物和相关试剂, 可直接在96孔板中操作完成, 具有快速、高通量、敏感性高、重现性好及廉价等特点。     

Estrogenic environmental chemicals and drugs: mechanisms for effects on the developing male urogenital system

Development and differentiation of the prostate from the fetal urogenital sinus (UGS) is dependent on androgen action via androgen receptors (AR) in the UGS mesenchyme. Estrogens are not required for prostate differentiation but do act to modulate androgen action. In mice exposure to exogenous estrogen during development results in permanent effects on adult prostate size and function, which is mediated through mesenchymal estrogen receptor (ER) alpha. For many years estrogens were thought to inhibit prostate growth because estrogenic drugs studied were administered at very high concentrations that interfered with normal prostate development. There is now extensive evidence that exposure to estrogen at very low concentrations during the early stages of prostate differentiation can stimulate fetal/neonatal prostate growth and lead to prostate disease in adulthood. Bisphenol A (BPA) is an environmental endocrine disrupting chemical that binds to both ER receptor subtypes as well as to AR. Interest in BPA has increased because of its prevalence in the environment and its detection in over 90% of people in the USA. In tissue culture of fetal mouse UGS mesenchymal cells, BPA and estradiol stimulated changes in the expression of several genes. We discuss here the potential involvement of estrogen in regulating signaling pathways affecting cellular functions relevant to steroid hormone signaling and metabolism and to inter- and intra-cellular communications that promote cell growth. The findings presented here provide additional evidence that BPA and the estrogenic drug ethinylestradiol disrupt prostate development in male mice at administered doses relevant to human exposures.     

CDRI-85/287: studies on competition to estrogen binding sites in the immature rat uterus.

Ability of compound CDRI-85/287, a new nonsteroidal antiestrogen with negligible inherent estrogenicity, to inhibit uptake of 3H-estradiol (3H-E2) by the immature rat uterus in vivo was investigated. Different doses of 85/287 were administered either intraperitoneally 30 min before 3H-E2 or orally 1 and 6 hr before 3H-E2. A dose dependent inhibition in 3H-E2 uptake was observed after administration of the compound by either route and was 69% at 50 micrograms/rat ip dose and 80% at 2.5 mg/kg po dose. In in vitro competitive binding assay, however, the compound showed poor affinity (RBA 0.42% of estradiol-17 beta) for cytosolic estrogen receptors. Considering the potent anti-estrogenic as well as anti-implantation efficacy of the compound, its action in vivo appears to be mediated via its active metabolite(s).     

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.     

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.     

Regulation of estrogen receptors in primary cultured hepatocytes of the amphibian Xenopus laevis as estrogenic biomarker and its application in environmental monitoring

The present study aims to introduce the regulation of estrogen receptors (ER) in primary cultured hepatocytes of the amphibian Xenopus laevis as a further potential estrogenic biomarker. Time courses of free ER in cell cultures treated with 17beta-estradiol (E2), nonylphenol (NP), and bisphenol A (BPA) were determined by means of radioreceptorassay (RARA). All compounds led to an immediate drop of free ER followed by a significant increase. The estrogen specific induction of ER-mRNA in vitro during time course was verified by using semiquantitative RT-PCR demonstrating greatest differences after 36 h. Dose-response curves of ER-mRNA for E2, NP, and BPA revealed that E2 possessed highest estrogenicity starting at 10(-9) M, while NP and BPA induced significant increases at 10(-8) and 10(-7) M, respectively. Extracts of the river Alb were subjected to RARA for ER binding to cytosolic liver fraction as well as to primary cultured hepatocytes for assessment of ER-mRNA induction. The results by RARA demonstrated clearly that binding to ER was highest in sewage treatment plant effluents and increased during the course of the river. These findings could be correlated with induction of ER-mRNA levels in vitro indicating that both techniques are suitable for application in monitoring of estrogenic EDC.     

Role of 17β-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17β-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17β-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17β-HSD isozymes have been characterized so far, and the data strongly suggests that different 17β-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17β-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17β-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17β-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17β-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17β-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17β-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Hormonal properties of norethisterone, 7alpha-methyl-norethisterone and their derivatives

Norethisterone (NET) is a progestagenic compound with very weak androgenicity and estrogenicity. These low androgenic and estrogenic activities may be attributed to NET itself or induced by metabolites of NET. In order to improve the bioactivity of NET, the effects of a 7alpha-methyl substitution were studied. Thus this study has two objectives: first the comparison between biological activities of NET and 7alpha-methyl-NET (MeNET), and second the biological activity of tentative metabolites of NET and those of MeNET. The metabolites consist of a 3-keto-, 3alpha- or 3beta-hydroxy-group located next to a carbon 4 to 5 double bond (Delta(4)) or a 5alpha-hydrogen atom. The 7alpha-methyl substitution was of special interest as it prevents 5alpha-reduction. The biological activities of NET, MeNET and their potential metabolites were assessed by in vitro binding, transactivation and proliferation assays on progesterone (PR), androgen (AR), estrogen (ER) and glucocorticoid (GR) receptors and by in vivo progestagenic McPhail, androgenic Hershberger, estrogenic Allen-Doisy tests and combined estrogenic and progestagenic ovulation inhibition tests. NET is a compound with five- to eight-fold weaker PR binding and transactivation activities than the reference compound Org 2058 (100%) and two-fold stronger than progesterone. Binding and transactivation activities of NET for AR (DHT=100%) are 3.2 and 1.1%, respectively, for ER none (E2=100%) and for GR below 1% (DEX=100%). MeNET is 1.5- to two-fold less progestagenic and ten- to 20-fold more androgenic than NET, while it does not show activity for ER and GR. The relative binding affinity of 5alpha-NET was seven-fold lower for PR and 1.5-fold higher for AR than for NET, while in transactivation assays 5alpha-NET was only active at levels below 1% for all tested receptors. 3beta-Hydroxy-(5alpha-reduced)-metabolites showed clear ER binding and transactivation activities, while 3alpha-hydroxy-(5alpha-reduced)-metabolites did hardly possess these characteristics. These hydroxy metabolites did not bind or activate other receptors. Substitution of 7alpha-methyl to NET metabolites led to similar characteristics, but with higher activities for AR and ER and weaker activity for PR. The outcome of in vivo tests showed a remarkable effect for MeNET. Progestagenic activity in rabbits appeared for NET equipotent to or eight-fold higher than for MeNET, after subcutaneous or oral treatment, respectively. On the other hand, MeNET showed in rats a ten-fold higher androgenicity and eight-fold higher estrogenicity than NET. Ovulation inhibition was induced at very low oral or subcutaneous dose levels, being 120- or ten-fold lower than for NET, respectively. The estrogenicity can also be induced by 3alpha- or 3beta-hydroxy metabolites of MeNET, which are 15 or even more than 40-fold stronger than those of NET, respectively. In conclusion, after the introduction of a 7alpha-methyl substituent to NET an increased estrogenicity and androgenicity and a reduced progestagenic activity was found. The in vivo estrogenicity is mainly due to 3beta-hydroxy-MeNET and to a lesser extent to 3alpha-hydroxy-MeNET, while the androgenicity and progestagenicity are most likely caused by MeNET itself. Since the 7alpha-methyl substituent inhibits 5alpha-reductase, 5alpha-reduced MeNET metabolites can be excluded from biological activities. As MeNET is a very effective ovulation inhibitor, due to its mixed progestagenic and estrogenic profile, a further reduction of androgenicity of MeNET may yield new contraceptives with an attractive profile for contraception.