Research Project
7302354
[unreadable] DESCRIPTION (provided by applicant): Genistein, a naturally occurring phytoestrogen found in soy, is a known in vitro activator of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR). In the genetic disease cystic fibrosis (CF) there is a loss of proper function of the epithelial CFTR chloride channel, resulting in dysfunctional epithelia, e.g. poor intestinal secretion. The mouse model for CF also exhibits an intestinal pathology (obstruction) similar to that in CF humans, requiring permanent use of laxatives for survival. Therefore there is currently a need for a treatment option to rescue this dysfunctional CFTR chloride channel in humans with CF. It is prudent to first observe an amelioration of defective CFTR function in the CF mouse model first. In normal wild-type (Wt) female mice, we tested intestinal function after 2 weeks of daily genistein injections (600 mg/kg body weight) and compared to genistein-free injected (vehicle control) mice. We found that female Wt mice injected with fed the genistein exhibited increased basal and cAMP- dependent chloride secretion compared to mice in the genistein-free group. These data are similar to our previous studies in females Wt mice fed dietary genistein for a period of 1 month. In addition, we have data to suggest that female CF mice fed the same genistein-containing diet have improved survival rates (88%). The goal of this proposal is to; (1) identify the effective genistein concentration to be injected (100, 300, or 600 mg/kg body weight) and the effective time period for the injection study (2 or 4 week duration), (2) identify the mechansim(s) involved in mediating the increased intestinal secretion with genistein-injected female Wt mice, and (3) to determine whether genistein-injected CF mice have improved survival rates. We hypothesize that daily genistein injections mediate an increased basal and cAMP-dependent secretion in female Wt mice, via activation of the CFTR channel. To further verify this we will inject a group of mice with an equivalent amount of daidzein (the inactive analog of genistein which has no effect on CFTR). We will utilize short circuit current measurements, to measure transepithelial secretion, and the patch-clamp technique on isolated single intestinal crypt cells to examine effects of genistein on single channel CFTR activity. In addition, after exposure to the injection protocols, we will examine in the intestinal epithelia; CFTR protein levels using Western Blot analysis, CFTR mRNA analysis using real-time RT-PCR, and effects on intracellular CFTR trafficking using immunofluorescent labeling of CFTR. Data from this project will provide information regarding potential usefulness of genistein as a therapeutic option in CF. This project aims to determine the mechanism by which genistein (a naturally occurring phytoestrogen found in soy) given as a daily injection over a period of 2-4 weeks to wild- type normal mice generates increased intestinal secretion. Furthermore, we aim to identify the suitability of genistein as a potential therapeutic for the most common lethal genetic disease in Caucasians, cystic fibrosis (CF). [unreadable] [unreadable] [unreadable]
