Estrogenic and genotoxic potential of equol and two hydroxylated metabolites of Daidzein in cultured human Ishikawa cells
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.
References (43)
Phyto-oestrogens and cancer
Lancet Oncol.
(2002)- et al.
Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)
Mut. Res.
(2003) - et al.
Rapid generation of homologous internal standards and evaluation of data for quantitation of messenger RNA by competitive polymerase chain reaction
J. Pharmacol. Toxicol. Meth.
(1997) - et al.
Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice
J. Nutr.
(2001) - et al.
Report from the in vitro micronucleus assay working group
Mut. Res.
(2003) - et al.
Induction of micronuclei. DNA strand breaks and HPRT mutations in cultured Chinese hamster V79 cells by the phytoestrogen coumoestrol
Food Chem. Toxicol.
(1997) - et al.
Oxidative metabolism and genotoxic potential of major isoflavone phytoestrogens
J. Chromatogr. B
(2002) - et al.
Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro
Chem. Biol. Interact.
(2004) - et al.
Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography
J. Chromatogr. B
(2003) - et al.
Overview of naturally occurring endocrine-active substances in the human diet in relation to human health
Nutrition
(2000)