Mammalian Cell DNA Damage and Repair Kinetics of

The rank order of genotoxic potency was IAA > BAA >> CAA from previous research. The concentration of each HAA was chosen to generate approximately th...
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Environ. Sci. Technol. 2009, 43, 8437–8442

Mammalian Cell DNA Damage and Repair Kinetics of Monohaloacetic Acid Drinking Water Disinfection By-Products Y U K A K O K O M A K I , †,‡ J U S T I N P A L S , ‡,§ ELIZABETH D. WAGNER,§ BENITO J. MARIN ˜ A S , †,‡ A N D M I C H A E L J . P L E W A * ,‡,§ Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, Center of Advanced Materials for the Purification of Water with Systems, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received June 23, 2009. Revised manuscript received August 26, 2009. Accepted September 8, 2009.

Haloacetic acids (HAAs) are the second most common class of chlorinated water disinfection by-products (DBPs). The single cell gel electrophoresis genotoxicity assay using Chinese hamster ovary (CHO) cells was modified to include liquid holding recovery time to measure genomic DNA damage and repair kinetics of three monoHAAs: chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA). The rank order of genotoxic potency was IAA > BAA >> CAA from previous research. The concentration of each HAA was chosen to generate approximately the same level of genotoxic damage. No cytotoxicity was expressed during the 24 h liquid holding period. Nuclei from CHO cells treated with BAA showed the lowest rate of DNA repair (t50 ) 296 min) compared to that of CAA or IAA (t50 ) 134 and 84 min, respectively). The different rates of genomic repair expressed by IAA or CAA versus BAA suggest that different distributions of DNA lesions are induced. The use of DNA repair coupled with genomic technologies may lead to the understanding of the biological and genetic mechanisms involved in toxic responses induced by DBPs.

Introduction The drinking water community provides an exceedingly important public health service by generating high quality, safe, and palatable tap water. Each day approximately 250000 public water purification facilities in the United States provide drinking water to 90% of the population (1). During the twentieth century, the outbreak of waterborne diseases, including typhoid and cholera, were greatly suppressed through disinfection (2). However, disinfectants react with natural constituents, e.g., organic matter, bromide, and iodide, in source water and form disinfection by-products * Corresponding author phone: 217-333-3614; e-mail: [email protected]. † Department of Civil and Environmental Engineering. ‡ Center of Advanced Materials for the Purification of Water with Systems. § Department of Crop Sciences. 10.1021/es901852z CCC: $40.75

Published on Web 10/02/2009

 2009 American Chemical Society

(DBPs); this unintended consequence may cause negative health effects (3). In order to reduce exposure, the U.S. Environmental Protection Agency (U.S. EPA) promulgated the Stage 1 Disinfectants and Disinfection Byproducts Rule (DBPR) in 1998 (4) and the Stage 2 DBPR in 2006 (5). In chlorinated water, haloacetic acids (HAAs) are the second most common class of DBPs (6). The sum of five HAAs (monochloro-, dichloro-, trichloro-, monobromo-, and dibromoacetic acid) is regulated at a maximum contaminant level of 60 µg/L (4, 5). The occurrence, genotoxicity, and carcinogenicity of HAAs were recently reviewed (7). All five regulated HAAs were mutagenic in bacteria (8) and induced genomic DNA damage in mammalian cells in vitro. Among the three monohaloacetic acids, iodoacetic acid (IAA) was the most cytotoxic and genotoxic, followed by bromoacetic acid (BAA) and chloroacetic acid (CAA) (9, 10). The rank order of their toxicity correlated with their electrophilic reactivity and the carbonhalogen bond dissociation energy (9). The rank order of teratogenic potency (neural tube developmental defects in mouse embryos) was IAA > BAA > CAA (11, 12). In general, brominated acetic acids were consistently more cytotoxic and more genotoxic than the corresponding chlorinesubstituted acids (7). Although there are many studies on the induction of DNA damage by DBPs, little information exists on the repair of DBP-induced DNA lesions (13, 14). In the literature, there is no systematic analysis of the DNA repair kinetics of regulated DBPs nor is there an example of correlating chemical structure activity relationships and repair. The aim of this research was to characterize the genotoxicity induced by monoHAAs in mammalian cells and determine the kinetics of DNA repair. We employed HAA concentrations that induced approximately equivalent genotoxic effects. Our hypothesis was that if DNA lesions induced by these HAAs were similar, then no statistical difference would be observed in the kinetics of DNA repair. If the different halogens (I, Br, and Cl) induced different DNA lesions, then the DNA repair kinetics would be significantly different.

Materials and Methods Reagents. General reagents were purchased from Fisher Scientific Co. (Itasca, IL) and Sigma Chemical Co. (St. Louis, MO). Bromoacetic (BAA) and chloroacetic acid (CAA) were purchased from Fluka Chemical Co. (Buchs, Switzerland). Iodoacetic acid (IAA) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Cell culture F12 medium and fetal bovine serum (FBS) were purchased from Fisher Scientific Co. The HAAs were dissolved in dimethylsulfoxide (DMSO) and stored at -22 °C in sealed, sterile glass vials. Chinese Hamster Ovary Cells. Clone 11-4-8 of the transgenic Chinese hamster ovary (CHO) cell line AS52 was used (15). The cells were maintained in F12 medium containing 5% FBS, 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA), and 1% glutamine and grown in 100 mm glass culture plates at 37 °C in a humidified atmosphere of 5% CO2. Treatment Conditions. The day before treatment 2 × 104 CHO cells were plated in 200 µL of F12 + 5% FBS medium per well in a sterile flat-bottomed 96 well microplate. The next day, the cells were washed with Hanks balanced salt solution (HBSS) and treated with the HAAs in F12 medium without FBS in a total volume of 25 µL for 4 h at 37 °C, 5% CO2. The wells were covered with AlumnaSeal. The concentrations of the HAAs for the DNA repair studies were first determined so that similar levels of genomic DNA damage VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. CHO cell viability throughout a 60 h time period with cells maintained under liquid holding conditions (F12 medium without FBS). were induced. The concentrations were 6 mM, 60 µM, and 25 µM for CAA, BAA and IAA, respectively. After treatment, the solution was aspirated, and the cells were washed two times with HBSS. The cells from one well were immediately harvested, and microgels were prepared for the determination of DNA damage with no time for repair. F12 medium without FBS (100 µL) was added to the other wells, and the microplate was returned to the incubator for designated times. This recovery period (liquid holding time) allowed for DNA repair. Single Cell Gel Electrophoresis. Single cell gel electrophoresis (SCGE or the Comet assay) quantitatively measures genomic DNA damage induced in individual nuclei of treated cells (16, 17) and has been used as a predictor of carcinogenic activity (18). The Comet assay is rapid, sensitive, and detects lesions, including DNA single strand and double strand breaks, incomplete excision repair sites, and alkali-labile sites. The assay combined with liquid holding allows for a quantitative analysis of DNA repair (13, 14, 17, 19, 20).

Detailed methods for preparation of SCGE slides and electrophoresis have been published previously (9, 10). After treatment, the cells were detached with 50 µL of a 0.005% trypsin solution in HBSS, followed by the addition of 70 µL of F12 + 5% FBS. An aliquot was analyzed for acute cytotoxicity with the trypan blue dye exclusion assay (21). The remaining cell suspension was embedded in a layer of low melting point agarose prepared in phosphate-buffered saline on clear microscope slides that were precoated with a 1% solution of normal melting point agarose prepared with deionized water. Two SCGE microgels were prepared from the suspension harvested from each well. The microgels were placed in lysing solution (2.5 M NaCl, 1% sodium lauryl sarcosinate, 100 mM Na2EDTA, 10 mM Tris, pH 10 with 1% Triton X-100, and 10% DMSO) overnight at 4 °C. The DNA of the nuclei was denatured for 20 min in electrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13.5). The SCGE microgels were electrophoresed for 40 min at 25 V and 300 mA (0.72 V/cm) at 4 °C. After neutralization with a 400 mM Tris buffer (pH 7.5), the microgels were dehydrated in cold methanol for 20 min, dried for 5 min at 50 °C, and stored at room temperature. For analysis, the microgels were hydrated in water at 4 °C for 20 min, and the nuclei were stained with 65 µL of an ethidium bromide solution (20 µg/mL) and rinsed in cold water. The microgels were kept at 4 °C and analyzed within a 4 h time period. Fifty randomly chosen nuclei per microgel were analyzed with a Zeiss fluorescence microscope (excitation filter of BP 546/10 nm, barrier filter of 590 nm) and a computerized image analysis system (Komet version 3.1, Kinetic Imaging, Ltd., Liverpool, U.K.). The digitalized data were automatically transferred from the CCD camera to a computer-based spreadsheet for statistical analysis. Statistical Analysis. Each experiment was independently repeated three times. The data were transferred to Excel spreadsheets (Microsoft Corp., Redmond, WA) and analyzed using SigmaStat 3.1, SigmaPlot 8.0 programs, and TableCurve 2D (SPSS, Inc. Chicago, IL). The primary measure of DNA damage and repair was the percent of the DNA that had migrated from the nucleus into the microgel (% tail DNA).

FIGURE 2. Comparative genomic DNA repair kinetics in CHO cells for chloroacetic acid (CAA), iodoacetic acid (IAA), and bromoacetic acid (BAA). 8438

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FIGURE 3. Histograms illustrating the distributions of SCGE % tail DNA values from CHO cells treated with bromoacetic acid (BAA), chloroacetic acid (CAA), and iodoacetic acid (IAA) without and with 3, 5, and 24 h liquid holding recovery.

TABLE 1. DNA Repair t50 Values of Genomic Damage Induced by IAA, BAA, or CAA haloacetic acid

treatment concentration

CAS number

t50 (min, sigmoidal regression)

t50 (min, first order regression)

IAA BAA CAA

25 µM 60 µM 6 mM

64-69-7 79-08-3 79-11-8

84 296 134

85 319 135

The distributions of the % tail DNA values are not normal. In order to conduct parametric statistical analysis the mean % tail DNA value was calculated for each microgel. The mean % tail DNA values for each experimental group were averaged. According to the central limit theorem, these averaged mean values were normally distributed (22); the data were analyzed using a two-way analysis of variance test statistic. The mean % tail value from the HAA-treated cells with no recovery time was set at 100% and compared with concurrently treated cells after designated periods of liquid holding. If a significant F value of P e 0.05 was obtained, a Holm-Sidak multiple comparison was conducted in order to determine if there was a significant difference in the rate of DNA repair among the monoHAAs.

Results and Discussion All three monoHAAs induced genomic DNA damage. In this study, the concentration of each HAA was selected to induce similar levels of genotoxic damage. The % tail DNA for each HAA at 0 time liquid holding was normalized to a value of 100%, which allowed for a direct comparison of the rates of DNA repair. Liquid Holding Time and Cytotoxicity. CHO cell viability was determined throughout a 60 h time period under liquid

holding conditions (F12 medium without FBS). More than 55 h of liquid holding time caused a decline in viability (Figure 1). There was no decrease in viability during the 24 h liquid holding period that was used for the DNA repair experiments. DNA Repair Kinetics. DNA repair kinetics was determined for IAA, BAA, and CAA, using SCGE and a liquid holdingprotocol (16, 17) (lower plot, Figure 2). The data for each DNA repair curve were regressed using curvilinear curve fitting as well as regression on the basis of first order kinetics (Table 1). The time required to affect 50% repair of the originally induced DNA damage (t50) was calculated by sigmoidal or first order regression analysis within the 0-24 h liquid holding time. With sigmoidal regression, the t50 values for IAA, CAA, and BAA were 84, 134, and 296 min, respectively (Table 1). The values were similar with first order regression. Using first order regression analysis, bimodal repair kinetics was observed. These bimodal repair curves may be due to the diversity of DNA lesions induced by the HAAs. Apurinic sites, single strand DNA breaks, and damaged bases can be repaired more efficiently and rapidly as compared to DNA cross-links and DNA double strand breaks. These latter lesions induce serious genomic damage and require more cellular processing for repair (23). The calculated first order repair rate (during the first ∼3 h of liquid holding) for IAA, CAA, and VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Two-Way Analysis of Variance for DNA Repair of CHO Cells Treated with IAA, BAA, or CAA (Holm-Sidak Pairwise Multiple Comparison) comparison CAA CAA BAA CAA CAA BAA CAA CAA BAA CAA CAA BAA CAA CAA BAA CAA CAA BAA

vs vs vs vs vs vs vs vs vs vs vs vs vs vs vs vs vs vs

IAA BAA IAA IAA BAA a IAA a IAA a BAA a IAA a IAA BAA a IAA a IAA BAA a IAA a IAA BAA IAA a

liquid holding time (h)

difference of means

P

0 0 0 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5

1.42 × 10-13 4.26 × 10-14 9.95 × 10-14 4.08 25.22 21.14 15.70 19.35 35.05 12.79 23.35 36.136 11.46 19.90 31.36 7.49 13.87 21.36

0.99 0.99 0.99 0.58 0.001 0.005 0.03 0.009 0.0001 0.08 0.002 0.0001 0.1 0.007 0.0001 0.3 0.06 0.004

a DNA repair levels were statistically different between the treatment groups at P < 0.05.

BAA was 0.842 h-1, 0.763 h-1, and 0.187 h-1, respectively. The rate of repair of BAA-induced lesions was different from the repair rates of CAA or IAA. Acute cytotoxicity was not observed throughout the liquid holding period (upper plot, Figure 2). Repair of DNA Damage Induced by Monohaloacetic Acids. Cells treated with BAA expressed a lower rate of DNA repair compared to IAA or CAA (Figure 2). To determine if there were significant differences in DNA repair at each liquid holding time, we used a balanced multivariant analysis of variance (ANOVA) with a Holm-Sidak pairwise multiple comparison test (Table 2). A significant difference in DNA repair kinetics was expressed by CHO cells exposed to BAA as compared to IAA or CAA. The DNA repair kinetics for CAA and IAA were statistically similar. Frequency Distribution of Repaired Nuclei. The levels of % tail DNA for each monoHAA as a function of liquid holding time were analyzed with frequency distribution histograms. Figure 3 illustrates the dynamics of genomic DNA repair in which the nucleus was the unit of measure. For each nucleus the distribution of DNA migration during each liquid holding time was plotted. The concentrations of monoHAAs were chosen to generate approximately similar levels of biological damage. The 0 h liquid holding time (Figure 3, far left panel of stacked plots) shows that CAA and IAA induced similar profiles of DNA damage. The damage induced by BAA with no liquid holding was much broader with fewer nuclei with the highest % tail DNA values but also many nuclei with lower amounts of damage. As the liquid holding time progressed from 3 to 24 h, the levels of DNA damage in each treatment group diminished, shifting the distributions to the left in each panel. However, the population of nuclei damaged by BAA showed less repair as compared to CAA and IAA (two center vertical panels). Few unrepaired nuclei remained after 24 h liquid holding (Figure 3, far right panel). DNA damage histograms for the untreated negative controls are presented in Figure 4; over a 24 h time period no significant difference was observed in DNA damage distributions. The level of completeness for the repair of DNA damage induced by each monoHAA can be observed by comparing the 24 h liquid holding distribution (Figure 3, far right panel) with that of the negative control (Figure 4). With no liquid holding time for repair, the mean of the % tail DNA was 60.5, 50.3, and 67.3 for IAA, BAA, and CAA, 8440

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FIGURE 4. Histograms illustrating the distributions of SCGE % tail DNA values from the CHO cell negative controls at 0, 5, and 24 h liquid holding recovery. No significant difference was observed among the three negative control groups (F2,1997 ) 2.81, P ) 0.06). respectively. After 24 h of liquid holding, the mean % tail DNA values were 7.5, 12.0, and 7.2, respectively. Liquid holding recovery significantly shifted the distributions to lower % tail DNA values; IAA showed the most rapid shift, CAA the next, and BAA the slowest (Figure 3). The mean % tail DNA values of the negative control did not change significantly: 4.8% without liquid holding, 4.1% after 5 h, and 4.3% after 24 h liquid holding recovery time (Figure 4). The different rates of genomic repair that are expressed by IAA or CAA versus BAA suggest that different DNA lesions and/or different distributions of DNA lesions are induced. Because the concentration of each haloacetic acid was chosen to generate approximately the same level of genotoxic damage, the null hypothesis was that there would be no difference in the DNA repair kinetics if similar DNA lesions were induced by the three haloacetic acids. The data do not support the null hypothesis. Currently there is not a complete analysis of the types of DNA lesions induced by the haloacetic acids. Cemeli et al. showed that the antioxidant butylated hydroxyanisole or catalase substantially reduced IAA-induced mutagenicity in Salmonella typhimurium and reduced genomic DNA damage in CHO cells (24). Oxidative stress may be a biological mechanism contributing to the genotoxicity of IAA. Oxidative stress has been linked with the toxicity of other DBPs, including 3-chloro-4-(dichlorom-

ethyl)-5-hydroxy-2(5H)-furanone (MX) (25), bromate (26-29), and chloroacetonitrile (30). Oxidative stress-induced DNA damage ranges from individual base damage to single strand and double strand DNA breaks (23). DNA repair of oxidative stress-induced lesions may be relatively fast if mediated by DNA glycosylases (31) or require more time if the lesion is a double strand DNA break (32). Recent toxicogenomic data indicate that noncytotoxic concentrations of BAA preferentially modulate the expression of human genes involved in double strand DNA (dsDNA) break repair (33). Double strand breaks are one of the most toxic mutagenic lesions and require more time for repair than other types of DNA damage (32). dsDNA breaks must be repaired to restore the integrity, stability, and reproducibility of the genome. XRCC3-241 is a mutant allele for dsDNA break repair; humans who carry this allele express higher risks of bladder cancer (34). It is intriguing that bladder cancer is associated with the consumption of disinfected drinking water (35). The use of DNA repair coupled with genomic technologies may lead to the understanding of the biological and genetic mechanisms that are involved in toxic responses induced by DBPs. Such knowledge may lead to the identification of biomarkers that may be employed in feed-back loops to aid water chemists and engineers in the overall goal of producing safer drinking water.

Acknowledgments This research was funded in part by Water Research Foundation Grant 4132. We appreciate the support by the Center of Advanced Materials for the Purification of Water with Systems, National Science Foundation Science and Technology Center, under Award CTS-0120978. The Heiwa Nakajima Foundation Fellowship Program is gratefully acknowledged for financial support to Y. Komaki.

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