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Testing the Carcinogenic Potential of Water Disinfectant Byproducts in a Human Colon Mucosal Culture System
Citation:
CHORLEY, B. N., T. Sheau-Fung, C. Jones, E. Winkfield, AND A. B. DEANGELO. Testing the Carcinogenic Potential of Water Disinfectant Byproducts in a Human Colon Mucosal Culture System. Presented at American Association of Cancer Research (AACR), Chicago, IL, March 31 - April 04, 2012.
Impact/Purpose:
Our overall results suggest the tested water DBPs may activate cellular mechanisms that mediate colon cancer development and etiology. Additionally, we demonstrate this in vitro methodology can detect carcinogenic potential of chemicals and may serve as a useful tool to test other chemicals in a more cost-effective and expedited manner than traditional animal bioassays.
Description:
Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer. Approximately 600 disinfection byproducts (DBPs) have been identified for a number of disinfectants currently in use. An in-depth mechanism-based structure activity analysis undertaken identified 50 unregulated DBPs with the highest risk carcinogenic potential. Because it would be financially prohibitive to test all priority DBPs in chronic animal bioassays, we are developing an in vitro model system to identify DBPs of carcinogenic potential in normal human colonocytes. Specifically, a mixed culture of human colon mucosal cells including stem cells with the capability for differentiation (NCM460) were treated with brominated DBPs bromochloroacetic acid (BCAA), dibromonitromethane (DBNM), and tribromonitromethane (TBNM), as well as a positive colon carcinogenic control, azoxymethane. To measure cancer-related pathways being activated, global gene expression analysis of 10-day 10-6M treated NCM460 monolayers was performed on Affymetrix Human Genome U133 Plus 2.0 arrays and statistically significant genes were identified using one-way ANOVA with a false discovery rate (Benjamini-Hochberg test) of ≤0.05 followed by a post-hoc test (Scheffe). Pathway analysis was performed using DAVID bioinformatics resource (National Institutes of Health) which identified multiple pertinent pathways activated with all or a majority of the treatments including cell adherins, ubiquitin-mediated proteolysis and xenobiotic metabolism. Of significance, AOM, DBNM and TBNM treatments induced genes involved in the WNT/ß-catenin, a well studied signaling pathway that modulates cell proliferation and differentiation and is a hallmark pathway for cancer development. As validation, immunocytochemical staining for ß-catenin indicated increased nuclear translocation after treatment with all DBPs and AOM when compared to the control. In addition, real-time RT-PCR analysis measured expression of select genes known to be directly regulated by ß-catenin after 7 and 10-day 10-6M AOM and TBNM treatment. MYC, AXIN2 and PPARD expression significantly increased (p<0.05, t-test) with 10, but not 7, day treatment with both chemicals, suggesting WNTß-catenin signaling pathway activation at the later time point. Importantly, preliminary studies indicate colony transformation of these cells after DBP treatment as measured in soft agar assay. Our overall results suggest the tested water DBPs may activate cellular mechanisms that mediate colon cancer development and etiology. Additionally, we demonstrate this in vitro methodology can detect carcinogenic potential of chemicals and may serve as a useful tool to test other chemicals in a more cost-effective and expedited manner than traditional animal bioassays. [Abstract does not necessarily reflect the policies of the U.S. EPA.]