The differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and proliferation in the rat is associated with a substance specific modulation of uterine gene expression

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Abstract

In this study the ability of the phytoestrogen genistein (GEN) to regulate proliferation in the rat uterine tissue and the associated molecular mechanisms were investigated in a dose and time dependent manner. A single administration of GEN induced a rapid increase of the uterine weight during the first 24 h. In contrast to E2, treatment with GEN for 3 days did not result in a further increase of the uterine weight. GEN only marginally effected the thickness of the uterine epithelium and the expression of epithelial proliferating cell nuclear antigen (PCNA). Whereas, estrogen sensitive genes were modulated significantly, the expression of key genes involved in the regulation of proliferation (PCNA, ERα /ERβ ratio) remained unaffected by GEN. Our results indicate that GEN has only a limited ability to activate molecular mechanisms involved in the induction of proliferation whereas estrogen sensitive genes are induced in a estrogen like manner.

Introduction

Over the last decades chemicals have been identified in a variety of plants that bind to the estrogen receptor (ER) and may trigger mechanisms of estrogen action (Davis et al., 1999). The most potent of these naturally occurring estrogens, also called phytoestrogens, are isoflavonoids, diphenolic chemicals detected in the bean subfamily of Leguminosidae. Even if the potency of most of these natural estrogens is low compared to endogenous or synthetic estrogens such as 17ß-estradiol (E2) or ethinylestradiol (EE), substantial quantities of these nonsteroidal estrogens are detectable in human urine (Adlercreutz et al., 1982). The molecular mechanisms involved in the action of these substances, their potential benefits or risks towards health issues are only barely characterised. Epidemiological data suggest that on one hand consumption of these environmental estrogens may have beneficial effects like protection against breast and prostate cancer (Adlercreutz et al., 1992, Haapiainen et al., 1988). On the other hand, there is the possibility that these compounds may act as endocrine disruptors, which might affect the endocrine system and may cause developmental (Setchell et al., 1987, McLachlan and Newbold, 1987) and reproductive disturbances (Setchell et al., 1984). Despite of all this lack in our knowledge, food or food supplements containing phytoestrogens are often being advocated as an alternative to hormone replacement therapy (HRT) in women with contraindications to the use of conventional estrogen replacement, or simply are in search for more ‘natural’ alternatives. Numerous studies have been performed with estrogenic plant extracts covering various aspects of the postmenopausal women’s health. Results have sometimes been conflicting and difficult to interpret (Russell et al., 2002). The lack of knowledge of what is the active ingredient precisely, its minimal effective doses, the lack of standardisation of the preparations used as well as the large individual variability of metabolism of precursors incorporated with the diet may all have played a role in confusing the issue about the effectiveness of these compounds.

More convincing are the data about the activity of purified phytoestrogens. The natural isoflavone phytoestrogen genistein (GEN) has been shown to stimulate osteoblastic bone formation, to inhibit osteoclastic bone resorption and to prevent bone loss in ovariectomised rats (Fanti et al., 1998). In addition, some studies demonstrate that GEN therapy improves endothelial function in postmenopausal women to a similar extent as does an estrogen/progestin regimen (Squadrito et al., 2003). Recent investigations have demonstrated that GEN and its derivates have the ability to act in a tissue specific manner thereby either behaving as a weak estrogen or as an anti-estrogen both in vivo and in vitro. Bound to the ER GEN evokes structural changes of this molecule comparable to selective estrogen receptor modulators (SERMs) like raloxifene (Pike et al., 1999). Several investigations in distinct in vitro and in vivo models have demonstrated that GEN modulates estrogen sensitive molecular parameters in a SERM-like manner (Diel et al., 2001a, Diel et al., 2001b). For all these reasons GEN may be referred as a Phyto-SERM. A potential benefit of SERMs in HRT is their ability to act in distinct estrogen sensitive tissues like the brain, the vascular tissue and the bone without affecting others like the breast or the uterine tissue. However, for some SERMs it has been demonstrated that their use is associated with unexpected and adverse side effects. The SERM Tamoxifen (Tam) for example acts like an antagonist in the mammary gland and for this reason it is used in breast cancer treatment since decades. In the bone, Tam acts like an agonist and contributes to maintain bone density. For these reasons, Tam in principle is a good candidate compound for HRT, but unfortunately it exhibits agonist-like activities in the uterus and stimulates proliferation of the uterine epithelium. Therefore, it was postulated that the use of Tam, especially in HRT, bears the risk of producing malignancies in the endometrium (Assikis et al., 1996). So far, the knowledge about the endometrial safety of phytoestrogens is very limited. For theses reasons we have investigated molecular mechanisms of the uterine activity of the phytoestrogen GEN in female ovariectomised rats. Special emphasis was given to the regulation of proliferation in the endometrium. Dependency of effects on dose, time and route of GEN administration were studied. The overall uterotrophic activity was correlated to the ability to induce proliferation and to modulate gene expression in the uterine tissue.

Section snippets

Substances

Ethinylestradiol (17α-ethinyl-1,3,5[10]-estriene-3,17β-diol;19-Nor-1,3,5[10],17α-pregnatrien-20yne-3,17-diol) was provided by the Schering AG (Berlin, Germany), 17-β estradiol (1,3,5[10]-estratriene-3,17β-diol) and genistein (4′,5,7-trihydroxyisoflavone) were provided by Sigma-Aldrich (Deisenhofen, Germany).

Animals

Juvenile female Wistar rats (130 g) were obtained from Moellegard (Moellegard breeding and research, Lille Skensved, Denmark) and were maintained under controlled conditions of temperature (20

Dose- and time dependent uterotrophic response after SC administration of GEN

The uterotrophic response to a single SC administration of three different doses of GEN (0.5, 2.5 and 10 mg/kg/BW) was assessed after 7 and 24 h. E2 (4 μg/kg/BW) and EE (4 μg/kg/BW) served as reference compounds. The uterine wet weight was evaluated. In Fig. 1A the gain of uterine wet weights following administration of increasing amounts of GEN is shown. The uterine wet weight was stimulated in a dose-dependent manner following treatment with GEN. A single SC injection of a dose of 2.5 mg/kg/BW

Discussion

Potential adverse or beneficial impacts of phytoestrogens on human health are controversially debated. Phytoestrogens, particularly GEN if given in early phases of life, are discussed as potential chemopreventive agents against breast cancer (Fritz and Cowardl, 1998, Lamartiniere et al., 1998). In addition, they seem to improve the condition of endothelial dysfunction (Squadrito and Altavilla, 2000), they are proposed to be protective against bone loss (Fanti et al., 1998) and more generally to

Acknowledgements

We thank Jan Seibel, Torsten Hertrampf and Angelika Friedel for careful and critical reading of the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft (DFG) grants: VO 410/6-3 and DI 716/9-1.

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