Elsevier

Toxicology Letters

Volume 158, Issue 1, 28 July 2005, Pages 72-86
Toxicology Letters

Estrogenic and genotoxic potential of equol and two hydroxylated metabolites of Daidzein in cultured human Ishikawa cells

https://doi.org/10.1016/j.toxlet.2005.02.011Get rights and content

Abstract

The soy isoflavone daidzein (DAI) is known to undergo metabolism to equol (EQO) and to 3′-hydroxy-DAI (3′-HO-DAI) and 6-hydroxy-DAI (6-HO-DAI) in humans. In order to better understand the implications of soy diets for human health, the hormonal and genotoxic activities of these DAI metabolites were studied in cultured human endometrial carcinoma cells. When the estrogenicity was tested by cell-free binding to recombinant human estrogen receptor (ER) α and β as well as by the induction of enzyme activity and gene expression of alkaline phosphatase (ALP) in Ishikawa cells, the ranking order was EQO > DAI > 3′-HO-DAI > 6-HO-DAI. All compounds had a higher affinity to ERβ than to ERα. No significant anti-estrogenic effects of the DAI metabolites were observed in the cells at non-cytotoxic concentrations. The in vitro genotoxicity was assessed by analyzing effects on cell cycle distribution and cell morphology as well as the induction of micronuclei (MN). EQO caused a slight increase in G1 and decrease in S phase of the cell cycle, and slightly but significantly induced kinetochore-positive as well as kinetochore-negative MN and an elevated proportion of abnormal mitotic spindles. 3′-HO-DAI, but not 6-HO-DAI, induced kinetochore-negative MN. The observation that major human metabolites of DAI exhibit estrogenic and genotoxic potential may be of relevance for the safety evaluation of diets containing soy isoflavones.

Introduction

There is substantial evidence from human epidemiological studies and from animal experiments that the isoflavones present in soy may have a beneficial effect on hormone-related neoplasia, e.g. cancer of the breast and prostate (Adlercreutz, 2002). On the other hand, some soy isoflavones have been reported to exhibit genotoxic potential in cultured cells (Munro et al., 2003). The major isoflavones present in soy are daidzein (DAI, Fig. 1) and genistein (GEN, Fig. 1), differing by just one hydroxyl group. GEN but not DAI acts as a clastogen in mammalian cells in vitro, indicating that small differences of the chemical structure can profoundly affect the biological activity of isoflavones. It has recently been shown that DAI is metabolized by rat liver microsomes to a variety of catechol metabolites (Kulling et al., 2000). The major in vitro metabolites of DAI were identified as 3′-HO-DAI, 6-HO-DAI and 8-HO-DAI (Fig. 1). These hydroxylated metabolites of DAI have also been demonstrated in incubations with human hepatic microsomes and in the urine of humans after ingestion of soy food (Kulling et al., 2002). In addition to hydroxylation, DAI is known to undergo biotransformation to equol (EQO, Fig. 1). This reductive metabolism is mediated by colonic bacteria and occurs in about one third to one half of all human individuals. EQO has been identified in human breast (Maubach et al., 2003) and prostate (Hong et al., 2002) tissue.

It was the aim of the present study to clarify the estrogenic as well as the genotoxic properties of the major DAI metabolites 3′-HO-DAI, 6-HO-DAI and EQO. The endogeneous estrogen 17β-estradiol (E2) and its 4-hydroxylated metabolite 4-HO-E2, which is believed to be involved in the mechanism of E2-mediated carcinogenesis (Liehr, 2000) were included in our study. The estrogenic and anti-estrogenic potential was determined at two in vitro endpoints, i.e. (1) the binding affinity to the human estrogen receptor-α (ERα) and β (ERβ) under cell-free conditions, and (2) the expression of the alkaline phosphatase (ALP) gene which was determined by mRNA quantification and measurement of enzyme activity. In vitro endpoints for genotoxic potential included (1) the induction of micronuclei (MN), (2) the effect on cell cycle distribution, and (3) the disruption of the cytoplasmic microtubule complex and the mitotic spindle. With the exception of ER binding, all endpoints were determined in cultured Ishikawa cells, a human endometrial adenocarcinoma cell line.

Section snippets

Chemicals

E2, 4-HO-E2 and DAI were obtained from Sigma (Taufkirchen, Germany), 3′-HO-DAI and 6-HO-DAI were purchased from Indofine (Somerville, USA), and 2,4,6,7-3H-E2 (89 Ci/mmol) from Amersham (Freiburg, Germany). All other chemicals, cell culture media and medium supplements were obtained from Sigma or Roth (Karlsruhe, Germany) if not specified otherwise.

Receptor binding assay

The affinities to recombinant human ERα and ERβ were determined according to Kuiper et al. (1998). Briefly, 200–300 pM recombinant human ERα or ERβ

Receptor binding

In order to assess the relative binding affinity of DAI and its metabolites to the human ERα and ERβ, the competitive displacement of 3H-E2 from the cell-free recombinant receptors by these compounds was measured in comparison with E2 and 4-HO-E2, whereas 4-HO-E2 bound with high affinity to both ERα and ERβ (Fig. 2), resulting in similar EC50 values (Table 1), the binding of DAI and its metabolites was markedly lower but showed a clear preference for ERβ over ERα (Fig. 2 and Table 1). In

Discussion

Soy food and soy-based nutritional supplements are currently gaining much popularity due to their putative beneficial health effects. In particular, the high content of the soy isoflavones GEN and DAI is believed to account for the protection against cancer of the breast and prostate as well as against cardiovascular diseases and osteoporosis (Mazur and Adlercreutz, 2000). Although the mechanisms of the protective effects are not completely understood, it is generally assumed that the

Acknowledgements

We thank Rosi Förster for helping with the ALP assay, Ling Jiang for the RNA isolations, and Margret Geller for helping with the flow cytometric measurements. We are also indebted to David Schumacher for valuable assistance in solving numerous laboratory problems.

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