Effect of dietary genistein on cell replication indices in C57BL6 mice
Introduction
Populations who consume a diet high in soy are reported to have a lower incidence of breast and prostate cancer. The activity of the soy-derived phytoestrogen, genistein, is thought to account for a portion of the chemopreventative properties of soy. The estrogenic properties of genistein have been well characterized and these properties contribute to the hormonal effects associated with soy consumption. However, the biological activities of genistein extend beyond its estrogenic properties and include its affinity for binding to the DNA synthesis-related enzyme, topoisomerase-II (topo-II) [1]. The binding of genistein to topo-II results in the stabilization of the cleavable complex that forms during DNA synthesis. A transient double-strand break, sequestered in the cleavable complex, is induced that allows the decatenation of the parental DNA and facilitates daughter DNA strand synthesis. Errors in the resealing of the transient double-strand breaks result from stabilization of the cleavable complex, and protein-linked DNA strand breaks, micronuclei, chromosome aberrations and mutations in the HPRT, TK and MLL genes have been detected in genistein-exposed cells [2], [3], [4], [5]. Further, the cell-cycle is altered in cells exposed to genistein as a result of both its topo-II-inhibitory properties and its activities as a tyrosine kinase inhibitor. Cells accumulate in S-phase due to the inhibition of DNA synthesis associated with cleavable complex stabilization [2] and in G2 as a result of the inhibition of phosphorylation at tyr-15 of the cyclinB/cdc2 complex [6], [7], [8], [9]. An increase in cells undergoing apoptosis also occurs in cells exposed to genistein. This may be due to a combination of genistein-induced tyrosine kinase inhibition (which signals cells into apoptosis) and the apoptosis signals from the DNA damage associated with the stabilization of the cleavable complex (reviewed in [10], [11]).
In order to extend these observations on the potential toxicity of genistein, a 28-day feeding study was conducted in the C57BL6 mouse. Both male and female animals were exposed to either a control diet or a diet containing one of five doses of genistein that were selected to encompass the range of human exposure (100–2000 ppm, based on dietary intake). Apoptosis and proliferation indices were determined in the three sections of the small intestine in order to serve as indicators of the potential toxicity of genistein. The small intestine was selected for analysis due to its high level of tissue replication and previous reports of an enhanced risk of azoxymethane-induced colon cancer in rats exposed to genistein [12]. Serum genistein levels were measured in peripheral blood samples in order to provide a measure of biological availability after exposure.
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Animals and animal husbandry
Six-week-old male and female C57BL6 mice were obtained from Taconic Farms, Germantown, NY. Animals were housed individually in polycarbonate cages with hardwood chip bedding. All procedures were approved by the NCTR Institutional Animal Care and Use Committee. Animals were held for a 14-day quarantine period, during which time the animals were acclimatized to an alfalfa- and soy-free diet (see below), and then assigned at random to treatment and control groups. Five animals per sex were
Serum isoflavone levels
Both total and aglycone (unconjugated) genistein levels were measured in serum obtained immediately before sacrifice. Serum concentrations of total genistein increased in a dose-responsive manner from below the detection limits (0.001 μM) in the control animals to 0.28 μM in males and 0.53 μM in females fed the 2000 ppm diet (Table 1). Serum aglycone levels also increased with increasing dose and were generally an order of magnitude less than the total genistein levels for each dose group (Table 1
Discussion
Our initial objective in these experiments was to determine the concentration of genistein in the serum of mice consuming doses of 100, 500, 1000, 1500 and 2000 ppm of genistein for 28 days. Based on actual food consumption and body weight measurements for the male and female C57BL6 mice used in this study, these diets delivered a daily dose of approximately 17, 90, 180, 270 and 360 mg/kg of body weight for males and 23, 115, 230, 345 and 460 mg/kg of body weight for females. These doses were
Acknowledgements
The authors would like to thank Dr Barry Delclos, Dr Sandra Culp and Ms Connie Weis for invaluable assistance in the design and conduct of these experiments. The critical reviews of Dr Delclos and Dr Robert Heflich are gratefully acknowledged.
This work was supported by an Interagency Agreement (FDA-224-93-001) between the National Center for Toxicological Research and the National Institute for Environmental Health Sciences. The author G.S. Akerman was supported by an Interagency Agreement
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