Estrogenic Activity of Griffonianone C, an Isoflavone from the Root Bark of Millettia griffoniana: Regulation of the Expression of Estrogen Responsive Genes in Uterus and Liver of Ovariectomized Rats

• Abstract
• Introduction
• Materials and Methods
• Chemicals
• Animals
• Uterotrophic assay
• RNA isolation and cDNA synthesis
• Real-time PCR
• Oligonucleotide primers for PCR reaction
• Statistics
• Results
• Discussion
• Acknowledgements
• References

Abstract

Griffonianone C (Griff C) is an isoflavone produced by Millettia griffoniana (Bail) and exhibits estrogenic properties in in vitro reporter gene assays. In order to validate its estrogenic potency in vivo, we administered subcutaneously Griff C (2, 10, or 20 mg/kg/d BW), 17β-estradiol (10 μg/kg/d BW) as positive control, and a vehicle control to ovariectomized Wistar rats. After three consecutive days of treatment animals were sacrificed 24 hours after receiving the last dose. The uteri and livers were excised, weighed and stored for mRNA expression analysis by real-time PCR. The uterine wet weight was not significantly increased by Griff C, although there was a trend towards an increase. In contrast, 17β-estradiol increased uterine wet weight 4.5-fold in comparison to the vehicle control. However, as revealed by real-time PCR Griff C affected the expression of estrogen-responsive genes in uterus and liver of ovariectomized rats. E2 induced a 550-fold stimulation of uterine C3 mRNA expression. Griff C at the dose 20 mg/kg/d BW caused a 50-fold up-regulation of complement C3 mRNA compared to the control. A significant increase in calcium binding protein 9-kilodalton mRNA expression was observed in the uterus of ovariectomized rats treated with E₂ (41-fold versus control) or 20 mg/kg/d BW of Griff C (25-fold versus control). In contrast, the expression of clusterin and progesterone receptor in the uterus was strongly decreased by both E2 and Griff C at the highest dose. We also found a repression of clusterin mRNA in the liver while carbonic anhydrase 2 and major acute phase protein were slightly up-regulated. In conclusion, Griff C showed a clear estrogenic action on uterine and hepatic tissues of ovariectomized rats, although its effect was less than the effect of estradiol. This suggests that some of the biological effects attributed to Millettia griffoniana are probably related to estrogen-mediated function.

Introduction

Many plant-derived bioactive substances with health benefits have attracted interest in the scientific community over the last two decades. Among these phytochemicals is the broad class of non-steroidal estrogens called phytoestrogens. Some of these substances like genistein and daidzein are known to bind to estrogen receptors (ER) and to induce estrogenic actions [1]. These health benefits which include alleviation of menopausal symptoms, limitation of bone resorption, and the lowering of cancer and cardiovascular disease risks may be due to the ER binding properties of certain plant derivatives. In Cameroon folk medicine crude extracts from root and stem bark of Millettia griffoniana (Bail) (M. griffoniana; Leguminosae) are used to treat boils, insect bites, sterility, amenorrhea as well as menopausal disorders, and illnesses with an inflammatory component like pneumonia and asthma. The main constituents of M. griffoniana have been determined to be diterpenoids [2], flavonoids and isoflavonoids [3], [4]. We [5] have previously shown that some isoflavones isolated from M. griffoniana exhibit estrogenic responses in different reporter gene systems expressing ERα.

The use of M. griffoniana in African folk medicine and its estrogenic responses in different reporter gene assays in vitro prompted us to further assess the estrogenic characteristics of griffonianone C (Griff C; Fig. [1]) as a compound isolated from M. griffoniana.

For primary in vivo studies the 3-day uterotrophic assay in rats or mice has been proven to be an efficient method for the determination of estrogenic activity [6]. Therefore, the main objective of this study was to use this assay for evaluating the estrogenic activity of Griff C in vivo. In the present study we applied the classical three-day uterotrophic assay in ovariectomized rats, one of the screening tests recommended for detecting and characterizing the estrogenic properties of potential endocrine disrupting chemicals [7]. We determined the uterotrophic responses of three different doses of Griff C and in addition evaluated its action on the regulation of estrogen-responsive genes in the uterus and liver.

Materials and Methods

Chemicals

17β-Estradiol (E2) was obtained from Sigma (Deisenhofen, Germany). Griff C was obtained as white crystals by repeated column chromatography over silica gel of the CHCl₃ extract of the ground root bark of M. griffoniana, and subsequent purification by preparative TLC (C₆H₆/petroleum ether/ETOAc, 3 : 6 : 1) [4]. Analysis of the ¹H-NMR and mass spectra of the tested compound showed a degree of purity of over 90 %.

Animals

Female Wistar rats (about 150 g) were obtained from Charles River (Charles River Wiga GmbH; Sulzfeld, Germany). Animals were housed in an environmentally controlled room (temperature 20 °C; humidity 50 - 80 %; 12-hour light/dark cycle). They were provided tap water ad libitum and a standard rat diet (SSniff R10-Diet; SSniff GmbH; Soest, Germany). All animal husbandry handling conditions were according to the Institutional Animal Care and Use Committee guidelines, regulated by the German federal law for animal welfare.

Uterotrophic assay

Wistar female rats were ovariectomized and allowed to recover for 14 days (endogeneous hormone decline). For the experiment, animals were randomly divided into five groups of six rats each. Rats were given daily subcutaneous injections of vehicle (control) or Griff C, 2 mg/kg/d BW; 10 mg/kg/d BW; 20 mg/kg/d BW in an ethanol-castor oil suspension (500 μL ethanol in 5 mL castor oil) for three consecutive days. As positive control for the effects of estrogen on uterine weight and gene expression, one group of animals was given daily injections of 10 μg/kg/d BW of E2. Animals were sacrificed 24 hours after the last administration by CO₂ inhalation after light anesthesia with O₂/CO₂ inhalation. Uteri and livers were collected and the uterine wet weight was determined. Uteri and liver tissues were frozen in liquid nitrogen for RNA preparation.

RNA isolation and cDNA synthesis

Total RNA was extracted from uterine or hepatic tissues (80 - 100 mg) using Trizol® Reagent (Life Technologies; Karlsruhe, Germany) according to the manufacturer’s protocol. The quality of RNA samples was examined on a 1 % agarose gel. DNA-free RNA was obtained by enzymatic treatment with deoxyribonuclease I (Roche; Mannheim, Germany) in the presence of ribonuclease inhibitor (Invitrogen; Karlsruhe, Germany) for 30 min at 37 °C. Equal amounts of total RNA from six rats in the same experimental group were pooled before cDNA synthesis.

For the first-strand cDNA synthesis, 3 μg of RNA together with 100 μg/mL oligo (dT)_{12 - 18} and 10 mM dNTPs (Life Technologies) were denatured at 65 °C for 5 min. The RNA was added to a mixture of reverse transcriptase buffer (manufacturer) containing 0.1 M dithiothreitol (DTT) (Invitrogen) and 200 U SuperScript® II reverse transcriptase (Life Technologies) in a final volume of 20 μL. The reaction was allowed to proceed for 60 min at 42 °C after which the enzyme was inactivated at 70 °C for 15 min.

Real-time PCR

Real-time PCR was performed using a Thermal Cycler with iQ real-time Detection System (BioRad; München, Germany). Each real-time PCR reaction consisted of 1 μL cDNA, 5 μL of 10 × buffer with 0.2 × SYBR Green I (Sigma-Aldrich; Taufkirchen, Germany), 2 - 4 mM MgCl₂ (Invitrogen Life Technologies), 0.2 mM dNTPs mix (Invitrogen Life Technologies), forward and reverse oligonucleotide primer mix (0.1 - 0.4 mM each), 10 nM Fluorescein Calibration Dye (BioRad), 0.5 U Platinum® Taq DNA polymerase (Life Technologies), and HPLC-treated water. After mixing, 50 μL aliquots of the mix were pipetted into each well of the 96-well thin-wall PCR plate (Bio-Rad). Reactions were carried out for 50 cycles (denaturation 95 °C for 10 sec, annealing at 60 °C for 15 sec, and elongation at 72 °C for 30 sec following an initial incubation for 3 min at 95 °C.). The reactions were run in triplicate.

Oligonucleotide primers for PCR reaction

The primers (MWG; Ebersberg, Germany) used for real-time PCR are listed in Table [1]. The relative mRNA amounts of target genes were calculated using the comparative cycle threshold method (ΔΔCT method) [8] with the mitochondrial cytochrome C oxidase subunit (1A) as endogenous reference gene, and relative to the negative control (probe from animals carrier treatment).

Statistics

The significance of the difference between control and treated groups was determined using ANOVA followed by a post-hoc multiple comparison (XLSTAT-Pro version 7.1, Addinsoft). The significance was determined at P values < 0.05, P values < 0.005, or P values < 0.001.

Results

The uterine wet weights in ovariectomized (OVX) rats following the administration of different doses of Griff C are shown in Fig. [2]. Griff C injected at 20 mg/kg/d BW to OVX rats produced a mild trend towards an increase of uterine weight, but this increase did not reach statistical significance, while E₂ at 10 μg/kg/d BW increased uterine weight 4.5-fold compared to control.

E₂ caused a significant reduction in the levels of both ERα and ERβ mRNAs (Fig. [3] A and B). In contrast to the effect of E₂ on the ERs expression, Griff C did not consistently alter the gene expression levels of ERα. A statistically significant effect was only detectable at the lowest dose. In addition it showed a clear down-regulation of ERβ gene at the dose 20 mg/kg/d BW and a slight, but statistically not significant up-regulation of the same gene at the two lower doses (Fig. [3] A and B).

To further assess the estrogenic effect of Griff C on the uterus, we examined the gene expression levels of complement C3 (C3), progesterone receptor (PR), and calbindin-9-kD (CaBP 9K), all carrying an estrogen responsive element (ERE) in their promoters. Each gene showed a unique pattern of regulation. Griff C at the dose of 20 mg/kg/d BW induced a significant up-regulation of the C3 mRNA levels. This increase was 49-fold in comparison to the carrier control but still less than the response to E2, which induced a stimulation > 500-fold (Fig. [4] A). Fig. [5] B shows the same data set without the E2 group to clearly illustrate the dose-dependency of the effect of Griff C on complement C3 expression. The CaBP 9K mRNA level was up-regulated by both E₂ and Griff C in the uterus (Fig. [4] C). E₂ could increase the CaBP 9K mRNA expression by more than 40-fold in comparison to control, whereas Griff C had no effect at low doses and its effect was almost half of that of E₂ at the dose 20 mg/kg/d BW.

As an example of a gene containing no ERE the regulation of expression of clusterin (CLU), a sensitive estrogenic response gene, was examined. A strong decrease of the uterine CLU gene expression was found in E₂ treated rats. A dose-dependent decrease was also observed in animals treated with Griff C (Fig. [5] A). Fig. [5] B shows a significant down-regulation of the PR, a gene with a complex promoter structure, mRNA expression in response to treatment of OVX rats with both E2 and Griff C, respectively.

We further examined the gene expression of cyclooxygenase II (COX 2) (Fig. [5] C) which was repressed in the uterus of OVX rats after treatment with E2. Griff C also induced a dose-dependent down-regulation of COX 2 mRNA levels.

The effects of estradiol and Griff C were not only assessed in the uterus, one of the major target organs of sex hormones but also in the liver. To study the hormonal regulation of expression of genes, we analyzed the expression of CLU, major acute phase protein (MAP), carbonic anhydrase II (CA2), and insulin-like growth factor binding protein (IGFBP1) in the liver of ovariectomized rats treated with E₂ or Griff C.

As observed in the rat uterus, the expression of CLU in the liver was also down-regulated due to the exposure to E₂ and Griff C (Fig. [6] A). In our study, IGFBP-1 was up-regulated by E₂. The results (Fig. [6] B) demonstrated a significant down-regulation of the expression of IGFBP-1 with almost 51 % reduction of the IGFBP-1 mRNA expression when OVX rats were treated with Griff C at the dose of 10 mg/kg/d BW. For MAP and CA2, an induction of the mRNA levels could be detected (Fig. [6] C and D), however this up-regulation was not statistically significant for all doses with regards to CA2.

Discussion

In this study, 17-β estradiol and Griff C were administered by a subcutaneous route at different doses selected on the basis of previous data from our laboratory [9]. Unlike treatment with E₂, Griff C did not result in an increase of the uterine wet weight. Griff C, however, was efficient in modulating changes in uterine complement C3 gene expression. This functional pattern is similar to that of the phytoestrogen genistein which induced a faint but statistically significant increase in the uterine wet weight only at a high dose and also stimulated complement C3 expression [10].

In order to gain potential mechanistic insights we investigated regulation of mRNA expression by Griff C on the ERα and ERβ levels. As previously described in the immature rat uterus [11] and mouse uterus [12], we also observed a reduction in the level of both ER-α and -β mRNA expression in the uterus of OVX rats after treatment with E₂ and Griff C. The adult uterus, which is a major estrogen target tissue, was found to have overall a very low expression of ERβ compared with ERα [13]. However, the lack of a full uterotrophic response to estrogen in female ERα knock-out mice suggests that ERβ plays only a minor role in the uterus [14]. The decline in uterine ERβ after 72 h of E₂ treatment occurs primarily in stromal cells [14]. This spatially links lower ERβ expression and estrogen action because estrogen-induced epithelial cell proliferation is mediated by ERs in stroma cells, not by those in the epithelial cells themselves [15].

We [5] have previously shown that some isoflavones isolated from M. griffoniana exhibit at high doses estrogenic effects in a reporter gene system expressing ERα. Since gene expression profiling can provide additional information on the estrogenic properties of chemicals, particularly towards their mode of action [16], [17] we additionally investigated the expression of several marker genes.

It was recently demonstrated that the down-regulation of CLU as well as the up-regulation of C3 gene expression are sensitive markers for estrogen action in the uterus [11], [18]. As expected, the expression of the C3 mRNA was strongly increased by E₂ while a significant reduction of the expression of CLU was found. We also observed that Griff C induced a dose-dependent down-regulation of CLU mRNA expression. This down-regulation of the CLU mRNA expression has been described in various steroid hormone-sensitive tissues like the uterus and the prostate [19]. While C3 is stimulated in both intact uterine tissue and tumor tissues, CLU is repressed in intact uterine tissue [20] and up-regulated in tumours [21]. These findings characterize the tissue-specific expression of some genes.

The synthesis of CaBP 9K in the rat uterus is estrogen-dependent in uterine stroma and smooth muscle tissue. During pregnancy it is also highly expressed in the uterine epithelium [22]. Estrogen effects on the expression of uterine CaBP 9K gene are due to direct transcriptional regulation through an imperfect palindromic estrogen-response element [23].

A strong correlation between effects of uterotropy and C3, CLU, and CaBP 9K mRNA expression was observed in E₂ treated rats, in contrast there was no relationship between the uterotrophic response to Griff C and its ability to modulate the expression of estrogen-sensitive genes.

PR expression plays a key role in the mediation of progesterone-induced actions and is modulated in the uterus by E₂ [24]. At all doses tested we observed that PR mRNA expression was down-regulated by both E2 as well as Griff C. These results corroborate previous findings [9], [25]; describing a decrease of PR mRNA expression in the uterus of ovariectomized rats after administration of E₂. Given the fact that in OVX rats not only is the production of endogenous E₂ disrupted but also the synthesis of progesterone, the reduction of the PR mRNA level is not due to the over-stimulation of PRs but is linked to ER and probably more closely related to ERβ than to ERα [26].

The COX 2 mRNA was strongly repressed in the uterus of OVX rats by both E2 and Griff C. These findings are contradictory to the results of Diel et al. [27] who found a strong induction of COX 2 in the uterus of OVX rats and a down-regulation in the vena cava of the same experimental animals. COX 1 and 2 are two rate-limiting enzymes involved in prostaglandin production [28].

Estrogens are capable of influencing several physiological functions in tissues with non-reproductive tasks like the liver. The expression of some hepatic proteins is regulated indirectly by estrogens via the modulation of pituitary growth hormone secretion [29]. Several other hepatic genes were shown to underlie direct regulation through the hepatic ER [30]. We have observed an up-regulation of IGFBP-1 mRNA induced by E₂ in the liver of OVX rats. IGFBP-1 is synthesized in the liver and is highly expressed in the decidualized rat uterus [31]. Previously, E₂ regulation of IGFBP-1 gene expression in liver and uterus had been demonstrated [32]. Its production in the liver was previously shown to be regulated by GH, insulin, dexamethasone, phorbol esters and glucagon [33], [34].

In conclusion, we found that Griff C, an isoflavone from Millettia griffoniana, has clear estrogenic properties; however, this action remains relatively low in comparison with the effects of the endogenous hormone E₂. Based on our results (and further planned investigations), the use of M. griffoniana products in African folk medicine to alleviate menopausal symptoms and menstrual disorders may be scientifically supported. Unlike phytoestrogens such as genistein, our results did not support the concept of a biphasic dose-response model characterized by the ability of a compound to induce stimulation at low-dose and inhibition at high-dose levels [35].

Acknowledgements

We thank S. Kolba and A. Beyer for technical assistance and David Riley (MD) for critical reading of the manuscript. G. Ketcha Wanda was supported by the German Academic Exchange Service (DAAD).

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Günter Vollmer

Technische Universität Dresden

Institut für Zoologie

Zellescher Weg 20b, Raum 253

01217 Dresden

Germany

Phone: +49-351-463-31922

Fax: +49-351-463-31923

Email: Guenter.Vollmer@tu-dresden.de

References

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  Search in Google Scholar
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Günter Vollmer

Technische Universität Dresden

Institut für Zoologie

Zellescher Weg 20b, Raum 253

01217 Dresden

Germany

Phone: +49-351-463-31922

Fax: +49-351-463-31923

Email: Guenter.Vollmer@tu-dresden.de