“Mechanisms of Genotoxicity of the Phytoestrogens Genistein and Daidzein”

DI VIRGILIO A.L.; BOLT H.M

Mainz, Alemania

Jornada; 44 Jornadas del DGPT (Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie).; 2003

The presence of hormonally active phytoestrogens in the human diet has been a matter of recent concern. Apart from the hormonally (estrogenic) activities of these compounds, the question of genotoxicity has been raised. Kulling and Metzler, 1997 have pointed to the possibility of differential genotoxicity of daidzein and genistein. The later differs from daidzein only by one additional hydroxyl group. The isoflavones genistein and daidzein were tested for cytotoxicity in order to find suitable concentrations for the Micronucleus (MN) assay. The Neutral Red uptake assay according to Riddel et al., 1984 was used to determine cytotoxicity. Chinese hamster fibroblast V79 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 and genistein was 25-150 µM and 5-100 µM, respectively. They were added as solutions of DMSO and incubated for 18 h. The ability to incorporate and bind Neutral Red and the total protein of viable cells were measured and compared with the DMSO control. Genistein and daidzein caused no overt cytotoxicity within the dose range investigated. The MN assay in V79 cells was used to confirm the preliminary data of Kulling and Metzler. V79 cells were grown in 25 cm2 flasks. Solutions of the test compounds in DMSO were added and incubated for 18 h. Following dissagregation with trypsin/EDTA and resuspension in medium, cells were subjected to hypotonic conditions with KCl and fixation. Cells were mounted on slides and stained with Acridine Orange. Genistein gave rise to a significant induction of micronuclei at concentrations of 5-25 µM. Daidzein showed a comparatively slight increase on the number of MN within the dose range of 25-100 µM. In summary, the differential genotoxicity of genistein and daidzein has been confirmed. Further studies will elaborate on mechanisms involved.