The uptake and delivery of genistein to the mammary gland and its action in mammary tissue are the focus of this project. The following questions are addressed in this application: 1) How are genistein and its glycosidic conjugates found in soy absorbed, metabolized, and then distributed into the peripheral blood compartment: these experiments will involve a) the study of intestinal uptake processes for each form of genistein, b) uptake and metabolism and excretion by the liver of genistein and its metabolites entering the portal circulation, and c) genistein metabolites in blood: in each of these areas of investigation, Dr. Barnes will examine the differences between orally administered (dietary fed) genistein and administration by injection, the dose-dependence of each of these processes, and the effects of chronic exposure to genistein. These studies will be conducted in female Sprague-Dawley rats using animals fitted with indwelling biliary cannulas. Intestinal absorption will be studied using everted intestinal sac preparations. Genistein and its metabolites will be identified and quantified using HPLC-mass spectrometry; 2) To what extent is genistein taken up by the cells of the breast? This question will be studied a) in vivo in the mammaries of rats exposed to genistein and its glycosidic conjugates, and b) in human mammary epithelial (HME) cells. The concentration of genistein in mammary glands dissected from the animals will be measured to determine if there is selective uptake of unconjugated genistein compared with its sulfate and glucuronide conjugates. In HME cells, the mechanism that accounts for the accumulation of genistein in these cells will be studied; 3) Does genistein upregulate TGFbeta1 (TGFb1)-dependent pathways in mammary cell lines? The effect of genistein on TGFb1 signaling, as documented by protein alterations for TGFb1 in other systems, will be examined in HME cells and a human breast cancer cell line, and in mammary glands dissected from rats exposed to genistein. In proposal #2 (entitled """"""""Dietary genistein for mammary cancer prevention"""""""") of this IRPG application, Dr. Lamartiniere will utilize the information gained in this study to optimize his investigations of the effects of dietary genistein administration on the prevention of mammary tumors in a rat model of mammary cancer.
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