Investigation on the Genotoxicity of the Isoflavones Genistein, Daidzein and Equol in V79 cells

DI VIRGILIO A.L.; BOLT H.M; DEGEN G. H.

Mainz, Alemania

Jornada; 45 Jornadas del DGPT (Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie); 2004

The presence of hormonally active chemicals in the human diet , such as man-made estrogenic or plant derived compounds (phytoestrogens), has become a matter of concern. A significant part of the human exposure to phytoestrogens is attributable to soy isoflavones. Apart from the hormonal activity of daidzein and genistein, the question of genotoxicity has been raised for these compounds. Equol is an important metabolite of daidzein, formed by the gut microflora. Equol is more estrogenic than daidzein, but its genotoxicity was unknown so far. Cytotoxicity of daidzein, equol and genistein was assessed with the Neutral Red uptake and BCA assay in Chinese hamster fibroblast V79 cells. Cells were seeded into a 96 well plate (104  cells/well in 200 µl DMEM medium) in the presence of different concentrations of phytoestrogens. The dose range for daidzein was 25-150 µM and for equol and genistein was 5-100 µM. The phytoestrogens caused no overt cytotoxicity within the dose range investigated. The alkaline elution (COMET) assay was used to detect DNA damage such as double and single strand breaks and alkali labile sites. When V79 cells were treated with isoflavones for 3, 12 and 18 h, a weak positive trend was observed for daidzein (25-100 µM), but not for genistein (5-50 µM) or equol for a 3 h treatment (5-50 µM). The micronucleus (MN) assay in V79 cells was then used to study chromosomal genotoxicity. V79 cells were grown in 25 cm2 flasks and treated with solutions of the test compounds in DMSO for 18 h. Cells were harvested, fixed on slides and stained with Acridine Orange for fluorescence microscopy. Genistein caused a clear dose-related induction of MN within the range of 5-25 µM. MN rates were declining at higher concentrations. By contrast, daidzein caused a comparatively shallow increase in the number of MN between 25-100 µM. Equol gave an intermediate response up to 50 µM. Additional staining with the anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST (-) micronuclei, i.e., MN containing chromosomal fragments, thus indicative of clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST (+) and CREST(-). Characterization of equol-induced micronuclei in under investigation. So far, our results confirm previous data on differential genotoxicity of genistein and daidzein and indicate a genotoxic potential for the isoflavone metabolite equol.