You are here:
Effects Of Haloacetic Acid Mixtures in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay
Citation:
NICHOLS, H. P., S. C. JEFFAY, M. BARRIER, M. G. NAROTSKY, J. Simmons, AND E. S. HUNTER. Effects Of Haloacetic Acid Mixtures in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay. Presented at Society of Toxicology Meeting, Salt Lake City, UT, March 07 - 11, 2010.
Impact/Purpose:
We have evaluated the effects ofHAA mixtures that represent those formed from disinfection of source water with low (LBM), medium (MBM) and high (HBM) levels of bromide.
Description:
The haloacetic acids (HAAs) are a class of chemicals produced as byproducts of drinking water disinfection. Source water characteristics (such as level of bromide) affects which HAAs are present in drinking water and their concentration. For example, high bromide-source water will contain higher concentrations ofbrominated-HAAs than low-bromide water. We have evaluated the effects ofHAA mixtures that represent those formed from disinfection of source water with low (LBM), medium (MBM) and high (HBM) levels of bromide. The proportions of HAAs in each mixture were determined from a previously published water chlorination model. The mixtures contained chloroacetic (MCA), dichloroacetic (DCA), trichloroacetic (TCA), bromoacetic (MBA), dibromoacetic (DBA), tribromoacetic (TBA), bromochloroacetic (BCA), bromodichloroacetic (BDCA) and dibromochloroacetic (DBCA) acids. The molar ratio ofthese compounds (MCA:DCA:TCA:MBA:DBA:TBA:BCA:BDCA:DBCA) in the LBM was 21:120:90:0:0:0.2:1:9:0.5. The molar ratio in the MBM was 14:60:32:8:43:11:61:40:49. The molar ratio in the HBM was 0:20:9:24:124:206:49:30:123. The Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to evaluate chemicals for effects on early differentiation and cytotoxicity. A single-cell suspension ofpluripotent 11 mouse embryonic stem cells (mESC) were plated in 96-well plates and maintained under differentiating conditions for 9 days. The concentration dependent effects of each mixture on cell number (DRAQ5/Sapphire700 staining) and differentiation (quantitative in-cell Western analysis of myosin heavy chain protein) were evaluated. The concentration that produced a 50% reduction (ED50) was calculated for each endpoint and reported for each mixture. The LBM produced effects on differentiation at 149X 93871-lM and altered cell number at 340X 21,420I-lM. In contrast, the MBM was potent at producing effects on cell number 29X 46.4I-lM, but did not alter differentiation at the highest testable concentration 1000X (> 1600I-lM). The HBM produced equal effects on differentiation and cell number at 77.2X 11271-lM and 76.2X 111OI-lM (respectively). Previously we reported, in whole embryo culture, MBM and HBM had similar potencies at producing dysmorphology and both were approximately 20 times more potent than LBM. The mESC assays showed that HBM was 10-20 times more potent than LBM for both cytotoxicity and differentiation. However, MBM was almost 25 times more potent than HBM in affecting cell number, but not differentiation. Thus, mixtures of haloacetic acids can affect mouse embryonic stem cells through alterations differentiation and proliferation or induction of excess cell death. [This abstract does not reflect EPA policy.]