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The Use of Normal Colon Cell Culture to Assess Toxicities and Cancer Molecular Pathway Alterations Induced by Disinfection Byproducts.
Deangelo, A., B. Chorley, S. Thai, C. Jones, M. George, S. Kilburn, E. Winkfield, AND M. Moyer. The Use of Normal Colon Cell Culture to Assess Toxicities and Cancer Molecular Pathway Alterations Induced by Disinfection Byproducts. Gordon Research Conference on Disinfection By-products, Mt. Holyoke College, MA, August 05 - 10, 2012.
Present a poster and abstract, at the Gordon Research Conference on Disinfection Byprouducts. Mt. Holyolk College, MA August 5-10-2012
Recent Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer (Bove, GE, Jr et al., Int. J. Health Geogr., 6:18, 2007). Approximately 600 disinfection byproducts (DBP) have been identified. Because it would be prohibitive to test even a small number of DBPs in chronic animal bioassays, we have undertaken the development of an in vitro model system to measure toxicities of individual DBPs and identify DBPs with a carcinogenic potential. Cell System: NCM460 cells, a mixed culture of human colon mucosal cells, are used. The cells stain positive for the epithelial cell markers, cytokertin, villin, and human secretory component and negative for endothelial cell and lymphocyte markers. The cells grow attached and in suspension and possess the capability for differentiation. Stem cells were identified using immunocytochemical staining for the markers, Lgr5+ and Musashi-1. The cells exhibit cytochrome P450 and GSTT1-1 activities comparable to those measured in the intact large intestine. Toxicity Tests: Approximately 50 DBPs (trihalomethane, haloacetic acid, halonitromethane, haloacetonitrile, and haloacetamide class) were tested for cytotoxicity in the growth inhibition assay (Plewa et al., Environ. Mol. Mutagen., 51:871, 2010). No significant correlation for the toxicity (IC50) between the colon cells and Chinese hamster ovary cells was observed for trihalomethane, haloacetic acid, halonitromethane, and haloacetonitrile DBPs. A strong correlation was found for the haloacetamide series. A strong correlation for the degree of toxicity of haloacetic acids between colon cells and mouse embryo cultures and mouse stem cells was also observed. Assays to detect DNA damage (comet assay, micronucleus assay) in colon cells are under development. Model system to identify DBPs with carcinogenic potential: NCM460 cells were treated with brominated DBPs (BCAA, DBNM, and TBNM), as well as a positive colon carcinogen, azoxymethane (AOM). 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 gene alterations were identified using one-way ANOVA with a false discovery rate of ≤ 0.05 followed by a post-hoc test. 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. Significantly, AOM, DBNM and TBNM treatments induced genes involved in the WNT/β-catenin, a well studied signaling pathway that modulates cell proliferation and differentiation and is a hallmark pathway for colon cancer development. As validation, immunocytochemical staining for β-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 β-catenin after 10-day 10-6M AOM and TBNM treatment. MYC, AXIN2 and PPARD expression significantly increased (p<0.05, t-test) for both chemicals. Importantly, preliminary studies indicate colony transformation of these cells after DBP treatment as measured in soft agar assay. We demonstrate this in vitro methodology can detect carcinogenic potential of chemicals and may serve as a useful tool to test other chemicals and mixtures in a more cost-effective and expedited manner than traditional animal bioassays. [This is an abstract or a proposed poster presentation and does not necessarily reflect EPA policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.]
Record Details:Record Type: DOCUMENT (PRESENTATION/ABSTRACT)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
INTEGRATED SYSTEMS TOXICOLOGY DIVISION