MECHANISMS OF INTESTINAL CARCINOGENESIS OF DISINFECTION

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A long term goal of our research program is to identify mechanisms that underlie genetic susceptibility to colon tumorigenesis induced by chemical carcinogens. In previous studies, we have examined the colon carcinogen, azoxymethane (AOM), which induces tumors in inbred mice and rats. In the following studies, we propose to evaluate the carcinogenic potential of an important drinking water disinfection bi-product (DBP), bromodichloromethane (BDCM). Using aberrant crypt foci (ACF) as an early biomarker, we will evaluate the ability of BDCM to induce malignant transformation within the intestinal epithelium of Fischer 344/N rats and C3B6F1 mice at early, intermediate and late time points after exposure through the drinking water. In order to assess malignant potential of colonic lesions, tissue sections containing ACF will be subjected to a complete morphometric analysis. To gain a further understanding of the mechanisms of tumorigenesis, tissue sections will be further subjected to immunohistochemical analyses using a panel of tumor biomarkers that are routinely used in our laboratory, including the following markers of cell proliferaton: PCNA, K-ras, cyclin D1 and CDK 4. In addition, we will evaluate the status of the following genes are associated with colon tumor progression, including p53, APC and beta-catenin. We will also measure cellular differentiation of colonoctyes using both dolichos biflorus agglutinin and hexosaminidase. It will also be important to assess the effects of BDCM on the rate of cell death within the colonic epithelium. We will therefore assay apoptotic response of carcinogen-exposed colon epithelium using commercially available reagents, including the TUNEL assay. Immunochemical analyses will be complemented by mutational analysis of candidate tumor-related genes, using PCR-SSCP analysis and direct DNA sequencing of cDNA prepared from freshly isolated tissue as well as samples procured by laser capture microscopy, techniques that are routinely used in our laboratory. To learn more about the potential role of these genes in the malignant conversion of BDCM-induced ACF, we will examine their mRNA expression using RT-PCR analysis. Depending on the outcome of our studies of BDCM, further analysis of related DBPs, including dibromacetic (DBA) acid, may also be undertaken. It is anticipated that our comparative morphometric and genetic analyses of target epithelial cell populations in rats and mice will provide important new insights into potential mechanisms by which DBPs induce precancerous lesions and tumors within the large intestine.