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Identifying Carcinogenic Potentials of Drinking Water Disinfection Byproducts using Normal Human Colonocyte Cultures
Citation:
DEANGELO, A. B., Y. Ge, M. George, C. Jones, S. Kilburn, S. Thai, W. Ward, AND E. Winkfield. Identifying Carcinogenic Potentials of Drinking Water Disinfection Byproducts using Normal Human Colonocyte Cultures. Presented at 5th International Environmental Science and Technology Conference, Houston, TX, July 12 - 16, 2010.
Impact/Purpose:
Report on the continued development of an in vitro/invivo assay for prioritizing unregulated DBPs.
Description:
Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer. Approximately 600 byproducts (DBPs) have been identified for the major disinfectants currently in use and represent less than half of the total organic carbon recovered. Because it would be financially prohibitive to test even a small number of the priority DBPs in chronic animal bioassays, we have undertaken the development of an in vitro/in vivo model system to identify DBPs with an ability to transform normal human colonocytes into malignant cells. Test System: NCM460 cells, a mixed culture of human colon mucosal cells, comprised the test system. The cells stained positive for the epithelial cell markers, cytokertin, villin, and human secretory component and negative for neural, endothelial cell and lymphocyte markers. The cells grew attached and in suspension and possessed the capability for differentiation, which indicated a stem-cell component. Stem cells were identified using immunocytochemical staining for the protein markers, Lgr5+ and Musashi-I. The cells exhibited cytochrome 450 and GSTTI-I activities comparable to those measured in the rat large intestine. Chemical Treatment and Cell Transformation: A preliminary qualitative dose-response experiment using the classical colon carcinogen azoxymethane (AOM) at IE +06 and IE +09, M (2: 90% viability) for 7 days has been completed. Following AOM removal, the cells growing in suspension were passaged into new flasks (S-cell fraction) and grown for another 7 days. The process was repeated with the passage of the Scells growing in suspension to generate the SI-cell fraction. The SI-cells were grown in soft-agar for 21 days. Visual observation showed the cells from the IE-06 and IE-09 M treatment groups formed colonies, a hallmark of cell transformation, in a dose-response manner. A quantitative dose-response experiment is underway using lE-06 -IE-12 M AOM. DBPs: Bromochloroacetic acid (rat colon carcinogen), dichloroacetic acid (rodent liver carcinogen), and two priority DBPs, dibromonitromethane (DNM) and tribromonitromethane (TBNM) were tested at IE+06 M (2:90%viability). The SI-fraction coloncytes from all treatments formed colonies in the soft agar. Genomic Changes: Analysis of gene expression intheSI-cell fractions fromallchemicaltreatmentsrevealedaltered expression ofthe Wnt/fJcatenin and adherens junction signaling pathways, which are critical for the development of colon cancer. Genomic analysis revealed a down-regulation of CtBP and TLE, repressor transcripts, and up-regulation ofTCF7L2 and TCF, transcripts that activate the down-stream Wnt target genes. Immunocytochemistry: Immunocytochemical methods confirmed the nuclear translocation of ~-catenin and a robust staining of the down-stream proteins, cyclin-D, c-jun and c-myc , which are essential for colonocytes to undergo oncogenesis. Summary: NCM460 cells retain many characteristics of colon crypt cells and can be transformed by drinking water DBPs. Genomic analysis and immunocytochemical staining demonstrated activation of the Wnt signaling pathway in a manner observed during the development of colon cancer in animals and humans. The growth oftransformed NCM460 colonocytes to form tumors in immunodeficient mice will used to verify the carcinogenicity of drinking water contaminants. Work on developing a high throughput assay is ongoing. The successful development of a rapid assay will be used to prioritize DBPs and complex DBP mixtures for further study. (This is an abstract of a proposed presentation and does not necessarily reflect the opinion ofthe US EPA.)