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Development of Normal Human Colonocyte Cultures to Identify a Carcinogenic Potential for Priority Disinfection Byproducts
Citation:
DEANGELO, A. B., C. JONES, M. H. GEORGE, S. Y. THAI, Y. GE, E. WINKFIELD, W. O. WARD, AND M. Moyer. Development of Normal Human Colonocyte Cultures to Identify a Carcinogenic Potential for Priority Disinfection Byproducts. Presented at 24th International Conference on Soils Sediments and Water, Amherst, MA, October 20 - 23, 2008.
Impact/Purpose:
To support MYP Drinking Water, LTG 1
Description:
Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer. Of the approximately >600 disinfection byproducts (DBPs) identified, the US EPA regulates 11 DBPS for an increased risk of cancer. An in-depth mechanism-based structure activity analysis undertaken to rank the carcinogenic potential of the >500 DBPs, identified 50 unregulated DBPs with the highest potential for carcinogenicity. We set out to develop an in vitro/in vivo model system to test the potential of unregulated DBPs to transform normal human colon cells and produce tumor growth. Two DBPs identified as rodent carcinogens, (bromochloroacetic and dichloroacetic acid), two priority chemicals (dibromonitromethane and tribromonitro¬methane), and the classic colon carcinogen, azoxymethane were selected. Human colon mucosal cells (NCM460) were exposed to 1 E+-06 M (≥90% viability) of each test chemical for 10 days. Following chemical removal, the cells (A fraction) were maintained in the growth media for 14-21 days. Cells growing in suspension (S fraction) were transferred to new flasks and grown for another 14-21 days. The process was repeated to generate an S2 fraction. These steps were necessary to deplete stem cells which have the capacity to grow in soft agar (Transformation Assay). The S1 fraction cells were grown in soft agar for 21-28 days. All of the treated S1 fractions formed colonies in the soft agar; S1 fractions from untreated cells did not form colonies. Colonies from each treatment were selected and grown out to provide cells to place into immunoincompetent mice (Tumor Formation Assay). Gene expression data generated from treated cells demonstrated gene activation in two pathways that were determined to be important to the development of colon cancer. The characterization and validation of this model continues.