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Immunocytochemistry and Image Analysis of Beta-Catenin Redistribution in Normal Human Colon Cell Cultures Treated with Disinfection By-Products.

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http://cfpub.epa.gov/si/si_public_comments.cfm

Citation:

WINKFIELD, E., M. Moyer, AND A. B. DEANGELO. Immunocytochemistry and Image Analysis of Beta-Catenin Redistribution in Normal Human Colon Cell Cultures Treated with Disinfection By-Products. Presented at The Society for In Vitro Biology Meeting, Charleston, SC, June 08 - 10, 2009.

Impact/Purpose:

This research represents an interesting technological approach to study and quantify chemical induced changes in cellular locations for important cellular molecules.

Description:

Epidemiological studies have shown an association between the consumption of chlorinated drinking water and increased risk for colon cancer. In vivo studies proved that rodents exposed to chlorination disinfection byproducts (DBPs) developed aberrant crypt foci (ACF) in the colon, and that some of these ACF developed into tumors. These ACF are characterized by altered Wnt signaling and changes in beta-catenin localization. We are developing an in vitro/in vivo assay to rapidly predict the carcinogenic risks posed by unregulated DBPs. The assay is based on the phenotypic transformation of NCM460 normal human colonocytes after exposure to test chemicals. Image analysis methods, using Alexa Fluor 488 conjugated antibodies to beta-catenin and DAPI DNA stain, were developed to characterize changes in the intracellular localization of beta-catenin after chemical exposure. These methods were used to analyze the intracellular relocation of beta-catenin in NCM460 colonocytes treated with the DBPs dibromo- and tribromonitromethane. We were able to demonstrate the relocation of beta-catenin from the plasma membrane to the nucleus in chemically treated colonocytes by means of fluorescence immunocytochemistry, image capture and analysis of colocalized events using Pearson's and Manders' coefficients and colocalization patterns. These methods provide a useful approach to identifying and assessing highly colocalized fluorescence activity generated by fluorescent antibody interactions with DNA staining.

Record Details:

Record Type:DOCUMENT( PRESENTATION/ ABSTRACT)
Product Published Date:06/10/2009
Record Last Revised:12/07/2009
OMB Category:Other
Record ID: 202867