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

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Abstract

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.

Introduction

The necessity to test chemicals for their potential estrogenic and antiestrogenic activities, has lead to the development of many estrogen receptor assays [1]. Most of these models use mammary carcinoma cells and measure growth potential, expression of endogenous genes or activation of reporter gene constructs as estrogenic endpoints [2], [3], [4], [5], [6]. However, it becomes more and more apparent that natural and synthetic compounds due to the potential complexity of the function two estrogen receptors exhibit an organ-selective mode of action [7]. Therefore it would be advantageous to be able to test environmental estrogens in organ specific in vitro models.

With this study we aimed to re-evaluate the activity of known xenoestrogens and to assess the potency of some new substances in the estrogen responsive human endometrial adenocarcinoma cell line Ishikawa derived from a well-differentiated endometrial adenocarcinoma [8], [9]. Alkaline phosphatase (AlkP) activity in these human endometrial cancer cells is markedly stimulated by estrogens [9]. In addition and in contrast to a yeast assays, which do not mimic human metabolism, the Ishikawa cells, like normal uterine cells, possess the important capacity to specifically metabolise female sex steroids and by conversion modulate their activity in uterine tissue [10], [11].

For the purpose of re-evaluation of environmental estrogens in an endometrial derived in vitro model we selected representative model substances of the three main classes of phytoestrogens, namely isoflavones, coumestrans and lignans [12], [13], [14] and well known environmental pollutants with estrogenic activity like bisphenol A, p-tert-octylphenol, nonylphenol and o,p′-DDT.

In addition, we aimed to contribute to the characterization of estrogenic activities of prenylated naringenins, which only recently became available in sufficient quantities and of potential estrogenic activities of plant extracts of the rhizome of black cohosh (Cimicifuga racemosa), which is used to treat menopausal complaints.

The three main classes of phytoestrogens comprise isoflavones, coumestrans and lignans [12], [14]. The major representatives of these classes are the isoflavones genistein and daidzein, which are found as β-glycosides in soy and other legumes. Coumestrol was also described as a potent estrogen in vitro [15], [16], [17], belongs to the coumestans. The sources of the coumestans are sprouts of mungo beans, soy beans, clover and alfalfa. Zearalanone is a metabolite of the non-steroidal estrogenic mycotoxin produced by Fusarium species [18].

8-Prenylnaringenin (8-PN), initially described as 8-isopentenyl naringenin, derived from heartwood of Anaxagorea luzonensis [19] has been characterized as a highly potent phytoestrogen [19], [20]. 6-(1,1-Dimethylallyl)naringenin (6-DMAN) contained in the leaves of an African tree (Monotes engleri) [21] has only recently been described for its estrogenic potential [22]. Both substituted naringenins are a lot more potent than the maternal substance naringenin, which is present in grapefruits (Citrus grandis). In fact, the activity of this compound is controversial debated in the literature [23], [24].

As representatives for environmental pollutants bisphenol A, alkylphenols and o,p′-DDT were selected. Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane) is used as a monomer to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances [25]. It was found as a contaminant not only in the liquid of the preserved vegetables but also in water autoclaved in the cans [26]. The widespread use of BPA has raised concerns about its effects in humans [27]. Alkylphenols (nonylphenol, NP; octylphenol, OP) are the final biodegradation products of alkylphenol polyethoxylates, which are non-ionic surfactant widely used in detergents, paints, pesticides, cosmetics and other formulated products [28], [29]. Alkylphenols are composed of an alkyl group that can vary in size, branching and position joined to a phenolic ring. o,p′-DDT is still widely used in several parts of the world to control malaria, typhoid and dengue vectors, even though its use was banned in many countries based on toxicity data in wild life species.

Finally, extracts from the rhizome of black cohosh Cimicifuga racemosa (CR) were tested as a representative of a medical remedy used for treatment of menopausal and post-menopausal disorders (for review see [30]). Again the estrogenic activities of these extracts are controversy discussed.

Overall several phytoestrogens, synthetic compounds with estrogenic activities and extracts from the rhizome of CR were found to exhibit as agonistic activities on AlkP in the endometrial Ishikawa model.

Section snippets

Materials

17β-estradiol, genistein, and zearalanone was purchased from Sigma, daidzein from Roth, coumestrol from Fluka, octylphenol and nonylphenol from Hüls, bisphenol A and o,p′-DDT from Aldrich.

The naringenins were synthesized as recently described [31] and provided to us by Prof. Dr. P. Metz (Department of Organic Chemistry, Dresden).

Ethanolic and isopropanolic extracts from the rhizome of Cimicifuga racemosa were prepared according to standard procedures and were provided by the manufacturer

Results

Ishikawa is an ER positive endometrial adenocarcinoma cell line derived from a glandular epithelial cell line. Applying realtime rtPCR the estrogen receptor-α but never the estrogen receptor-β could be detected in Ishikawa cells. These cells respond estrogens and antiestrogens at concentrations approximating between 10−10 and 10−5 M. The responses have therefore to be regarded as pure estrogen receptor-mediated.

Using a modified in vitro bioassay test systems in 12 well microtiter plates we

Discussion

It is well known, that estradiol and several other estrogens enhance the activity of alkaline phosphatase (AlkP), an enzyme known to be regulated by ovarian hormones in the non-pregnant and pregnant rodent and monkey uterus [32]. The effect was inhibited by antiestrogens like 4-hydroxy-tamoxifen [9], [33]. This property of the Ishikawa cells was often used to test potential estrogenic or antiestrogenic properties of compounds by several bioassay test systems with Ishikawa cells [1], [9], [34],

Acknowledgements

We are grateful for the expert technical assistance provided by S. Kolba, M. Wulf, and A. Beyer. The authors also wish to thank the group of P. Metz for syntheses of the naringenin and the derivatives. This paper in part was supported by the Deutsche Forschungsgemeinschaft (Vo410/6-1) and by the State of Baden-Württemberg (BWplus 99015a).

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