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Development of normal human colon cell cultures to identify priority unregulated disinfection by-products with a
A B DeAngelo1, C P Jones, M P Moyer
1National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, NC 27711, USA. deangelo.anthony@epa.gov
Abstract:
Research was initiated to develop an in vitro system to identify disinfection by-products with a potential to transform normal human colonocytes into malignant cells. Tribromomethane and bromochloroacetic acid, rodent colon carcinogens, dibromonitromethane and tribromonitromethane, recently identified in drinking water, and azoxymethane, a classic colon carcinogen, were tested for the ability to transform NCM460 cells. The chronic toxicity was determined for the series of trihalomethanes, haloacetic acids and halonitromethanes as well as NCM460 cell enzymatic capabilities. The order of cytotoxicity was halonitromethanes > haloacetic acids > trihalomethanes. Cytotoxicity within a series increased with the degree of bromination and decreased with the molecular weight. The genotoxicity profile was similar to that for cytotoxicity. Enzymatic analysis demonstrated that NCM460 cells possess glutathione-S transerase-1-1 and CYP450 activity similar to that measured in the large intestine. NCM460 cells were exposed to 10(-6) M of the test chemicals for three days. While NCM460 cells from all treatments had the ability to grow in soft agar to some extent, only cells exposed to azoxymethane or tribromomethane were able to grow in media lacking serum and growth factors. When sub cultured, NCM460 cells exposed to 10(-9) M azoxymethane for three weeks formed colonies with morphology distinct from untreated cells.
Insights
Researchers developed an in vitro system to test drinking water disinfection by-products for colon cancer-causing potential. Halonitromethanes were most cytotoxic, and azoxymethane and tribromomethane showed cell transformation abilities.
Area of Science:
- Environmental Health
- Toxicology
- Cell Biology
Background:
- Disinfection by-products (DBPs) in drinking water are a public health concern.
- Some DBPs are known or suspected carcinogens.
- An in vitro model is needed to assess DBP carcinogenicity.
Purpose of the Study:
- To establish an in vitro system for identifying DBPs that can transform normal human colonocytes into malignant cells.
- To evaluate the cytotoxicity and genotoxicity of specific DBPs and a known colon carcinogen.
- To assess the enzymatic capabilities of NCM460 cells for relevance to in vivo studies.
Main Methods:
- NCM460 human colonocyte cells were exposed to azoxymethane, tribromomethane, bromochloroacetic acid, dibromonitromethane, and tribromonitromethane.
- Chronic toxicity and enzymatic capabilities of NCM460 cells were determined.
- Cells were assessed for anchorage-independent growth (soft agar) and growth in serum-free media.
- Morphological changes in subcultured cells were observed.
Main Results:
- Cytotoxicity order: halonitromethanes > haloacetic acids > trihalomethanes.
- Cytotoxicity increased with bromination and decreased with molecular weight within chemical series.
- Genotoxicity profile mirrored cytotoxicity.
- NCM460 cells exhibited glutathione-S-transferase-1-1 and CYP450 activity.
- Azoxymethane and tribromomethane induced significant cell transformation, including growth in serum-free media and distinct colony morphology.
Conclusions:
- The in vitro system effectively identified DBPs with carcinogenic potential.
- Halonitromethanes represent a significant cytotoxic and genotoxic risk among tested DBPs.
- Azoxymethane and tribromomethane demonstrated potent transforming abilities in NCM460 cells, warranting further investigation.
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