Project Account Number: R/WF-09-06
PRINCIPAL INVESTIGATOR: Michael J. Plewa
INITIATION DATE: March 1, 2006
COMPLETION DATE: February 29, 2008
AFFILIATION: University of Illinois at Urbana-Champaign

Michael J. Plewa
University of Illinois
364 National Soybean Research Center
1101 W Peabody
Urbana, IL 61801
Phone: 217-333-3614
mplewa@illinois.edu

Objectives
The goal of this research is to generate an in vitro mammalian cell chronic cytotoxicity and acute genotoxicity database that will focus on priority DBPs and related compounds. This research will develop a comparative database and will link the analytical chemistry and analytical biology of the priority DBPs identified in the Nationwide Occurrence Study. Most of the priority DBPs are not commercially available, however, we have access to small quantities (50 mg) of these agents that were synthesized for EPA for use as analytical chemical standards. The mammalian cell cytotoxicity and genotoxicity database will serve as a practical resource for the water treatment community for use in their decisions on disinfection practice. This research problem is covered by IISG topic area Water for the Future, “Develop a better understanding of the fate and effect of toxic chemicals….” The objectives of this research proposal are to, (i) select EPA priority haloamide, haloketone DBPs and N-nitrosamine DBPs, (ii) conduct a chronic mammalian cell cytotoxicity analysis on these DBPs, (iii) rank-order the DBPs based on their cytotoxicity and compare them to our current published database and to positive control toxicants, (iv) determine the genomic DNA-damaging capacity of each DBP in mammalian cells, and (v) rank-order each DBP for mammalian cell genotoxic potency and compare to our current published database and to positive control carcinogens.

Methodology
We propose employing Chinese hamster ovary (CHO) cells in microplate-based assays that we developed and calibrated, with support from the U.S. EPA and AwwaRF for the analysis of toxic responses of DBPs and other hazardous chemicals. Short-term assays have limitations in the breadth of the phenomenon measured. However, they provide depth and with sufficient numbers of replicates a precise statistical basis for comparative toxicology. We propose to use two endpoints, (i) a chronic cell cytotoxicity assay and (ii) the measurement of acute genomic DNA damage. The microplate cytotoxicity assay measures the reduction in cell density as a function of DBP concentration over a period of ~3 cell divisions (72 h). Chronic cell cytotoxicity is a very useful measurement of the biological effects of low concentrations of DBP over several cell generations. The endpoint will be expressed as the %C1/2 value which is the concentration of the test agent, determined from a regression analysis of the data, that reduces the cell density to 50% of the concurrent negative control. The single cell gel electrophoresis (SCGE) assay is very sensitive and can quantitatively determine genomic DNA damage and predict carcinogenic potency. We will expose CHO cells to specific DBP concentrations for 4 h; the cells will be washed, embedded in layers of agarose prepared with an electrolyte and the cell membranes will be lysed. The nuclei will be electrophoresed and the amount of damaged genomic DNA will be quantitatively measured by its migration in the gel. A computerized image analysis system will be used to measure various SCGE parameters of the evaluated nuclei. The digitalized data will be automatically transferred from the CCD camera to a computer-based spreadsheet for statistical analysis. A genotoxic potency value will be calculated for each DBP. The data will be compared with known positive control standards as well as with our DBP database.

Rationale
By 2030 the population in the 16 county region of southeastern Wisconsin, northeastern Illinois and northwestern Indiana is projected to increase by 2.3 millions people or over 21%. Lake Michigan currently supplies drinking water for over 10 million people. With this research we have the opportunity to expand our current research program to develop a quantitative toxicity database on priority drinking water disinfection by-products (DBPs). Each year approximately 250,000 public water purification facilities in the United States provide over 4.7x1012 liters of high quality drinking water to 90% of the population. In Illinois over 11 million people rely on public water treatment facilities for drinking water. The production and distribution of disinfected water was a profound public health triumph of the twentieth century that significantly reduced waterborne diseases. Disinfection of drinking water primarily employs oxidant chemical disinfectants that convert naturally occurring organic material along with bromide and iodide in the source water into chemical disinfection by-products (DBPs). While reducing the public health risk of acute infection by waterborne pathogens, the unintended generation of DBPs poses a chronic health risk. DBPs represent an important class of environmentally hazardous chemicals that carry long-term human health implications. Epidemiological studies demonstrate that individuals who consume chlorinated drinking water have an elevated risk of cancer of the bladder, stomach, pancreas, kidney, colon and rectum as well as Hodgkin’s and non-Hodgkin’s lymphoma. DBPs have been linked to adverse reproductive and developmental effects. Approximately 600 DBPs have been identified; this represents only a fraction of the halogenated organic material that is isolated after the disinfection of raw waters. Although most research focuses on the public health aspects of exposure to DBPs there are unrecognized environmental consequences of disinfection water. Each year in the United States approximately 2.6x10⁶ kg of toxic halogenated compounds are generated and released as a result of water disinfection. The impact of this toxic stress upon the environment is unknown. Recently the U.S. EPA carried out a Structure-Activity Relationship (SAR) analysis to identify a set of priority DBPs. Using this information the EPA conducted the Nationwide Occurrence Study and generated a list of priority DBPs. Unfortunately there is little toxicity information on these priority drinking water DBPs. It is the goal of this research to correct this problem.