Drinking Water Disinfection Byproduct Iodoacetic Acid Induces

May 6, 2013 - (3-5) Many emerging DBPs induce higher levels of cytotoxicity, mutagenicity, ... Sixty milliliter water samples were collected in headsp...
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Drinking Water Disinfection Byproduct Iodoacetic Acid Induces Tumorigenic Transformation of NIH3T3 Cells Xiao Wei,†,‡,∥ Shu Wang,†,∥ Weiwei Zheng,†,∥ Xia Wang,† Xiaolin Liu,† Songhui Jiang,† Jingbo Pi,§ Yuxin Zheng,Δ Gengsheng He,*,⊥ and Weidong Qu*,† †

Key Laboratory of the Public Health Safety, Ministry of Education, Department of Environmental Health, School of Public Health, Fudan University, Shanghai, 200032, China ‡ Department of Occupational and Environmental Health, School of Public Health, Guangxi Medical University, Nanning 530021, China § Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709, United States ⊥ Key Laboratory of the Public Health Safety, Ministry of Education, Department of Nutrition and Food Hygiene, Fudan University, Shanghai, 200032, China Δ National Institutes of Occupational Health and Poison Control, Chinese Centers for Disease Control & Prevention, Beijing 100050, China ABSTRACT: Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 μg/L IAA and 0.86 μg/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 μM exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 μM). IAA, but not IF, induced a concentration-dependent DNA damage measured by γ-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern.

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chlorinated and brominated analogues.8,12,13 Plewa et al.14 demonstrated that iodoacetic acid (IAA) was more mutagenic, genotoxic, and cytotoxic in Salmonella typhimurium and mammalian cells than bromoacetic acid or chloroacetic acid. In mammalian cells, including human cells, IAA is the most cytotoxic, genotoxic, and mutagenic DBP among haloacetic acids (HAAs).8,15 The cytotoxicity and genotoxicity of IAA in mammalian cells14,16 was 100 times higher than that of 3chloro-4-(dichloromethyl)-5-hydroxy-2-(5H)-furanone (MX), the most mutagenic DBP observed in S. typhimurium.17 Similarly, iodoform (IF) is the most cytotoxic among iodotrihalomethanes (THMs)8 and is more toxic than regulated THM4. Thus, IAA

INTRODUCTION Drinking water disinfection is essential to protect the public from waterborne diseases and represents a major public health achievement of the 20th century; however, toxic disinfection byproducts (DBPs) unavoidably formed in the production of drinking water are a public health concern. Accumulating evidence indicates that exposure to DBPs is associated with bladder and colon cancer1,2 and may induce adverse reproductive and developmental effects.3−5 Many emerging DBPs induce higher levels of cytotoxicity, mutagenicity, genotoxicity, and teratogenicity compared with regulated DBPs.6−9 The potential adverse effects of these unregulated DBPs have increased the concern of scientists, government officials, and the public. Iodinated DBPs (I-DBPs) are a class of emerging toxic DBPs.10 Initially, I-DBPs in drinking water were an issue because of taste and odor.11 I-DBPs were shown to be generally more cytotoxic or genotoxic in mammalian cell assays than their © XXXX American Chemical Society

Received: November 22, 2012 Revised: April 26, 2013 Accepted: May 6, 2013

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microplates at 37 °C for 24 h. Subsequently, different concentrations of IAA or IF were added into the wells and incubated further for 72 h. The control cells consisted of only medium for calibration. The concentrations that are lethal to 50% of cells (LC50) were determined from analysis of the log− linear phase of the curves. Cell Colony Formation Assay. Colony formation was determined by the method of Dunkel et al.36 NIH3T3 cells seeded into a 6-well plate at a density of 100 cells/well were cultured for 24 h. Subsequently, the cells were treated with IAA, IF, 0.5% dimethyl sulfoxide (DMSO)/distilled water (negative control), or 3-methylchloanthrene (positive control) for 72 h. At the end of the treatment, the medium was removed, and the cultures were rinsed three times with PBS. After rinsing, fresh growth medium was added to the plates. The cells were then cultured for 7 days, and the medium was changed twice a week. On day 7, the cell colony was fixed and stained. Only colonies consisting of more than 50 cells were included in colony counts. The colony-forming efficiency (CFE) and relative colonyforming efficiency (RCFE) were calculated as follows: CFE (%) = (number of colonies formed/number of cells seeded) × 100%; RCFE (%) = (CFE on treated plate/CFE on negative control plate) × 100%. Determination of DNA Damage. DNA damage was determined by γ-H2AX phosphorylation assay37 and single cell gel electrophoresis (SCGE).38 H2AX is a variant isoform of the histone H2A protein that is expressed in chromatin. In response to DNA double-strand breaks (DSBs), serine 139 in its unique carboxy terminus is phosphorylated rapidly by DNA damage checkpoint kinases to form γ-H2AX. Expression of γ-H2AX was used as an early biomarker for DNA DSBs. NIH3T3 cells were treated with different concentrations of either IAA or IF for 24 h, with cell survivorship of >75%.39 Three replicate plates were prepared for each concentration of DBPs. Hydrogen peroxide (H2O2) and distilled water/DMSO were used as positive and negative controls, respectively. Expression of γ-H2AX was quantified using a γ-H2AX phosphorylation assay kit with the supplier’s protocol (Catalog no. 17-344) (Upstate, Lake Placid, NY) and analyzed directly on a FACS Calibur cytometer (BD Biosciences, San Jose, CA). In the SCGE assay, the harvested cells were embedded in an agarose microgel and lysed; the DNA was denatured and electrophoresed under alkaline conditions (pH 13). The SCGE tail moment was measured by an CASP image analysis system (Comet Assay Software Project Lab).40 The experiments were repeated three times. Cytokinesis-Block Micronucleus (CBMN) Assay. The CBMN assay was conducted following the methods of Fenech41 and the Organization for Economic Co-operation and Development.42 Briefly, NIH3T3 cells were exposed to different levels of IAA or IF for 40 h (1.5−2 normal cell cycles), with the highest concentration selected to produce 55% ± 5% cytotoxicity. Three replicate plates were prepared for each concentration of DBPs. Mitomycin C and distilled water/DMSO were used as positive and negative controls, respectively. Two thousand binucleated cells were scored per concentration, and the induction of micronuclei (MN) was calculated according to the criteria described by Fenech.41 The experiments were repeated three times. Cell Transformation. A cell transformation assay was carried out as described previously.36 Briefly, NIH3T3 cells were seeded at a density of 2000 cells per flask. After 24 h, the cells were treated with IAA, IF, 3-methylchloanthrene (positive control) or DMSO (negative control) for 72 h. The concentrations chosen

and IF are recognized as important I-DBPs in drinking water because of their toxic characteristics. However, IAA and IF as well as other I-DBPs are not regulated by the World Health Organization, the United States Environmental Protection Agency (U.S.EPA), or the Chinese Ministry of Health. Only IF is on the list of priority DBPs.18 The formation of I-DBPs in drinking water depends on the iodine concentration in the source water and the disinfection process. The ground and surface source waters of coastal cities may contain high levels of bromide and iodide which is caused in part by salt water intrusion. Shanghai is a classic coastal city, with the Huangpu River and Yangtze River serving as its main water sources. As for many drinking-water utilities in the United States, to reduce THM4 and HAA5 and meet stricter regulations, water plants in Shanghai have changed disinfection practice from chlorine to chloramines. Chloramination reduces the formation of total organic halogen (TOX); however, it promotes the formation of total organic iodine (TOI),19−21 I-DBPs,13,22,23 and N-nitrosamines.24,25 The possible health effects of I-DBPs, especially IAA and IF, require additional biological evaluation based on their potent cytotoxicity and genotoxicity.10,13,14,26−31 There are no data on the carcinogenicity of IAA available either in vitro or in vitro. IF lacks a 2-year or whole-life rodent bioassay, although a 19.5 months study was negative.10 Therefore, the objectives of this study are to measure the occurrence of IAA and IF in the drinking water of Shanghai and determine the cytotoxicity, genotoxicity, and potential carcinogenicity of IAA and IF in mammalian cells with a battery of analytical bioassays.

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MATERIALS AND METHODS Reagents. All chemicals were purchased from Sigma (St Louis, MO) unless specified otherwise. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were obtained from Invitrogen (Grand Island, NY). Sampling and Determination of IAA and IF Concentration. Samples of Shanghai raw and finished drinking water were collected from two plants that used the Yangtze River as their raw water and two plants that used the Huangpu River as their raw water in January (salt water intrusion period) and July (no salt water intrusion period) of 2011. Sixty milliliter water samples were collected in headspace-free amber glass containers with polytetrafluoroethylene-lined screw caps containing quenching agents. Samples were extracted within 2 h and stored at 4 °C until analysis. IAA and IF were detected by gas chromatography with electron capture detection (GC/ECD, Shimadzu 2010) as described previously.32,33 Cell Culture. The NIH3T3 cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM containing 10% FBS and 100 U penicillin/mL at 37 °C in a humidified 5% CO2 atmosphere.34 Animals. Balb/c nude mice were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Science, and maintained with controlled temperature (22 ± 2 °C), humidity (50%), and light/dark cycle (12/12 h). The animal treatment protocol was approved by the Animal Ethics Committee of Fudan University. Cytotoxicity Assay. Cell viability was evaluated using a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5(2,4disulfophenyl)-2H-tetrazolium monosodium salt] assay kit (Dojindo, Kumamoto, Japan) as detailed previously.35 One thousand NIH3T3 cells per well were seeded in 96-well B

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chloraminated drinking water from regions containing high levels of iodide in their water source.13,14,18 As shown in Figure 1A and B, the concentrations of IAA and IF in drinking water

ranged from the maximal nontoxic concentration to a level at which 80−90% toxicity was observed. Subsequently, the cells were rinsed with PBS (pH 7.4) and cultured with growth medium without the toxicants for 10 days. The medium was refreshed every 3 days. On day 10, the measurement of transformed foci was confirmed by 10% Giemsa staining, and the transformation frequency (TF) was calculated as follows: TF = [total number of transformed colonies per treatment/(total cells plated per treatment × CFE)] × 100%. The CFE comes from the cell colony formation assay. In addition, the transformed cells resulting from 5 μM IAA exposure were used for a concanavalin A agglutination assay, soft agar growth assay, and assessment of tumorigenicity in nude mice. The normal, untransformed cells taken from the untreated plates were used as the negative control. Concanavalin A Agglutination. The concanavalin A agglutination assay was conducted as detailed by Rottmann et al.43 Concanavalin A is a plant lectin that specifically binds to sugars at cell surface macromolecules, which is increased in transformed malignant cells. The attachment of the single cells to the cell layer is determined by the concentration of concanavalin A addition or incubation time. Compared with normal cells, malignant cells are agglutinated easily in a short time or with a low concentration of concanavalin A. The transformed cells from transformed foci on day 10 were harvested and adjusted to 104 cells/mL with PBS (pH 7.4). A series of concentrations of concanavalin A and 100 μL single cell suspensions were added to 24-well microplates and gently mixed for 10 min. The progression and rate of cell agglutination with concanavalin A were observed by microscope (Olympus, Japan). Soft Agar Assay. The soft agar growth assay followed the method detailed by Booden et al.44 A 3 mL aliquot of 0.6% agar in culture medium was plated in 60-mm dishes. One thousand cells from transformed foci were mixed with 3 mL of 0.35% agar in medium and plated on the solidified bottom agar. When the top agar solidified, the dishes were transferred to an incubator and cultured for 30 days. Each dish was fed with 2 or 3 drops of complete growth medium every 2−3 days. The colonies were counted on day 30. Tumorigenicity in Nude Mice. The tumorigenicity of transformed cells was investigated by the method of Engle and Schou.45 The IAA-transformed cells were suspended and adjusted to 1 × 107 cells/mL with PBS (pH 7.4), then 0.2 mL of the cell suspension was subcutaneously injected in the right lateral aspect of the Balb/c nude mice. The mice were inspected for tumor growth three times per week. Tumor size was measured in two perpendicular dimensions (longest × shortest2 diameter × 0.5). After 30 days, the mice were autopsied immediately and inspected. Tumors were fixed in buffered formalin for pathological examination. Statistical Analysis. Data were analyzed using the GraphPad Prism software (version 5.0, GraphPad Prism Inc., San Diego, CA). A one-way analysis of variance test was conducted to determine statistically significant differences among treatment groups. If a significant F value (P < 0.05) was obtained, a Dunnett multiple comparison versus the control group analysis was applied. The statistical tests were two-tailed with significance levels of 0.05. Results were expressed as mean ± standard deviation (SD).

Figure 1. Concentration of IAA and IF in drinking water at four water plants. (A) IAA was found in drinking water from four water plants, at a maximum level of 2.18 μg/L and minimum level of 0.03 μg/L. (B) IF was found in drinking water from four water plants at a maximum level of 0.86 μg/L and was not detected in drinking water of water plants using the Yangtze River as source water in summer.

were related to water source and demonstrated a seasonal variation. IAA was detected in drinking water from the Huangpu River and Yangtze River in both winter and summer but concentrations were 7- to 9-times higher in winter than in summer. IF was found in the Huangpu River and Yangtze River water plants during the winter and Huangpu River plants at lower levels in the summer. The concentrations of IAA and IF were higher in the drinking water from the polluted Huangpu River than those from the Yangtze River. The maximum concentrations were 2.18 μg/L for IAA and 0.86 μg/L for IF in drinking water from the Huangpu River. Cytotoxicity of IAA and IF in NIH3T3 Cells. As shown in Figure 2A and B, IAA and IF were cytotoxic in NIH3T3 cells as measured by the WST-8 assay and colony formation assay. IAA showed cytotoxicity at a concentration as low as 2.5 μM, whereas IF was cytotoxic above 55 μM. The LC50 of IAA and IF were 2.77 ± 0.15 and 83.37 ± 0.99 μM, respectively, demonstrating that IAA has potent cytotoxicity. These results are consistent with those reported by Plewa et al.8,14,46 DNA Damage Induced by IAA and IF in NIH3T3 Cells. To investigate the genotoxicity of IAA and IF in NIH3T3 cells, DNA damage as induced by these two compounds was determined using the γ-H2AX phosphorylation assay and SCGE. As shown in Figure 3A and B, both IAA or IF exposure (24 h) resulted in a significant increases in γ-H2AX expression. The induction of γ-H2AX was concentration-dependent for IAA but not for IF. These results demonstrated that IAA and IF were able to damage DNA and could induce double strand breaks. In

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RESULTS The Occurrence and Concentrations of IAA and IF in Shanghai Drinking Water. I-DBPs were discovered in C

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Figure 2. Cytotoxicity of IAA and IF in NIH3T3 cells. Cells were treated with (A) IAA or (B) IF for 72 h. Cell viability was measured by WST-8 assay (left axis) and the cell colony forming assay (right axis). Data are shown as mean ± SD of three independent experiments.

addition, both IAA and IF caused an increase in the SCGE for the tail moment in a concentration-dependent manner (Figure 3C and D). In contrast, IAA and IF did not increase the frequency of micronuclei in NIH3T3 cells (Figure 3E and F). Transformation of NIH3T3 Cells by IAA but Not IF. The cell transformation assay is a method to detect and quantify in vitro malignant transformation of cells; positive results are viewed as predictors of in vivo carcinogenesis.47 IAA induced the formation of transformed foci seen at day 10 and produced a concentration-dependent increase in transformed foci (Figure 4A). The lowest effective concentration of IAA that induced a significant increase in transformed foci was 2 μM. However, IF was negative in the transformation assay (Figure 4B, C). The positive control 3-methylchloanthrene induced 17.0 ± 2.5 transformed foci per dish and a TF of 3.81%. Transformed cells are often associated with certain phenotypic changes, such as agglutination with concanavalin A, anchorage independence (cells form colonies in soft agar), and tumorigenicity. These assays were applied to determine the characteristics of transformed cells. As shown in Table 1, nontransformed NIH3T3 cells were agglutinated only with a high (100 μg/mL) concentration of concanavalin A, whereas IAA-transformed cells were agglutinated with 25 μg/mL concanavalin A. Negative control cells failed to grow when suspended in soft agar, whereas IAA-transformed cells grew in soft agar (not shown). The average frequency ± SD of transformed colonies was 13.47 ± 0.87%, significantly higher than the control. An important proof of the tumorigenicity of transformed cells is to observe their ability to form progressively growing tumors in immunologically nonresponsive animals.42 The NIH3T3 cells that were transformed with 5 μM IAA produced tumor nodules after transplantation into nude mice. By 15 days after transplantation, tumors were evident and increased in size in all nude mice injected with IAA-transformed cells (Figure 4D). No tumor growth was observed after injecting negative control cells (Table 2). The mean ± SD of tumor size was 3252.2 ± 158.2 mm3, and the 95% confidence interval (CI) for the mean was 2894.2−3610.1 mm3 (Table 2).

Figure 3. Determination of γ-H2AX phosphorylation, SCGE and micronucleus formation induced by IAA or IF in NIH3T3 cells. Expression of γ-H2AX in NIH3T3 cells exposed to (A) IAA or (B) IF for 24 h. The tail moment induced by a 24 h-exposure to (C) IAA or (D) IF was measured by SCGE assay. Binucleated cells with micronuclei after exposure to (E) IAA or (F) IF for 40 h. Data were pooled from three independent experiments. Data are shown as mean ± SD. An asterisk (*) indicates P < 0.05 vs control.

Histologic examination of the tumors revealed that the cells were arranged diffusely or overlapped in fascicles with intratumoral hemorrhage and necrosis. As shown in Figure 4E and F, the tumors were poorly differentiated fibrosarcoma without a tumor capsule. Tumor cell morphology was spindle-shaped with minimal cytoplasm and a large spherical or irregular nucleus with dark blue color. Mitotic figures and abnormal mitoses were frequently observed. These results strongly suggest that IAA is a potential carcinogen.

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DISCUSSION Sea water intrusion, which increases the formation of DBPs, is a challenge for drinking water production in estuary water sources around the world. Halogen salts in seawater lead to an increase in the more toxic I-DBPs formed after drinking water disinfection. The genotoxicity of IAA indicated that it might be a potential genotoxic carcinogen,10,13,14,26,27 and the findings concerning D

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Figure 4. IAA-induced transformation of NIH3T3 cells. (A) Representative images of normal (left) and IAA-transformed cells (right). (B, C) Transformation frequencies induced by (B) IAA and (C) IF are shown. (D) Growth curves of tumors that formed in nude mice by subcutaneous injection of IAA-transformed cells. Tumor size (longest × shortest2 diamater × 0.5 in mm3) is plotted against time. Each line represents an individual animal, n = 10. (E and F) Representative histologic images of a tumor formed in nude mice (H and E. staining). Hemorrhage and necrosis were present in tumor tissue (E). Mitotic figures and abnormal mitoses were frequently observed (F).

Table 1. Concanavalin A Mediated Agglutination in IAATransformed NIH3T3 Cells

Table 2. Tumorigenicity of IAA-Exposed Cells after Subcutaneous Injection into Nude Mice

concanavalin A concn (μg/mL) a

cells

0

12.5

25

50

100

control IAA-treated

− −

− −

− +

− +

+ +

cellsa control IAAtreated

a

no. of mice with tumorb/no. of mice (%)

tumor size (mm3, 95% CI)

0/10 (0) 10/10 (100)

0 3252.15 (2894.2−3610.1)

Control represents the normal, untransformed cells; IAA-treated represents transformed cells that developed after exposure to 5 μM IAA for 72 h. “−” indicates negative result. Cells were dispersive. “+” indicates positive. Cells agglutinated together.

Control represents the normal and untransformed cells; IAA treated represents the transformed cells that had been exposed to 5 μM IAA for 72 h. bTen mice were subcutaneously injected with cells in each group. After 30 days, mice were autopsied immediately and inspected.

mutagenicity and genotoxicity of IF were inconsistent among different test end points.28−31,48 However, there are no 2-year or

the whole-life rodent carcinogenicity data on IAA and IF available.10 The present study demonstrated that IAA is an in

a

E

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anchorage-independent growth in soft agar, and tumorigenicity in nude mice.36 In this study, IAA induced malignant transformation in NIH3T3 cells. These data demonstrated that IAA was a potent inducer of NIH3T3 cell transformation with a positive response at 2 μM (3.7 × 10−4 mg/mL). This is a significant finding and underscores the importance of the possible adverse health impacts of the I-DBPs. Humans may be exposed to low concentrations of IAA in their drinking water and over a lifetime. On the basis of the strong cytotoxicity and genotoxicity of IAA, long-term and low-dose exposure may have an adverse impact on public health. No long-term rodent carcinogenicity data and no human epidemiological information are available for IAA exposure in human populations. This study confirmed past research that IAA was a potent cytotoxic and genotoxic agent in mammalian and human cells. IAA at low concentration induced malignant transformation in the NIH3T3 murine cell line. Our data expand the information on IAA as a potential serious hazard to the public health. Future studies are needed to combine toxicology with determination of IAA, the molecular mechanisms of action, and human epidemiology to generate a comprehensive assessment of the adverse effects of IAA on public health and the environment.

vitro carcinogen, on the basis of prolonged exposure of NIH3T3 cells to IAA resulting in tumorigenic transformation and formation of fibrosarcoma after transplantation into nude mice. IAA and IF for the first time were identified in the drinking water of Shanghai, China. The formation of IAA and IF was related to seawater intrusion into the source waters and disinfection with chloramination, as previous studies reported.13,18,22,23 Although the concentrations of IAA and IF in Shanghai drinking water are at low-microgram-per-milliliter levels, they are the most potent cytotoxin in the HAA and THM families.8,46 The toxic potency was governed by the halogen leaving group and the relative alkylation potential of the monoHAAs.46 In this study, the tested LC50 values for IAA (2.77 μM) and IF (83.37 μM) in NIH3T3 cells were similar to the previous studies in CHO-AS52 cells (2.95 and 66.00 μM, respectively), indicating that the chronic toxicity of both cell lines was not different.8,13,46 Previous studies indicated that several levels of genetic damage were induced by I-DBPs.13,26−31,46 IAA produced positive results in the Ames test, CHO-K1/hypoxanthine−guanine phosphoribosyl transferase gene mutation assay, and comet assay, but it was negative for induction of MN in binucleated cells.14,26,27 IF was negative in the comet assay, and it induced reverse mutations in various S. typhimurium strains in the Ames test.28−30 Here, we found that IAA induced DNA strand breaks in NIH3T3 cells with the sensitive comet and γ-H2AX assays. These findings further supported the earlier results.14 However, IAA and IF did not induce MN formation in binucleated NIH3T3 cells. Liviac et al. also reported that IAA did not increase the micronucleus frequency in binucleated TK6 cells.26 These differences may be accounted for by DNA repair kinetics.49 The γ-H2AX and comet assays detect primary DNA damage that may be efficiently repaired, whereas the CBMN test monitors DNA damage that escapes repair and induces chromosome breaks. However, IAA could cause a high level of modulation in the expression of DNA DSB repair genes involved in BRCA1, BRCA2, and ATR pathways. These toxicogenomic studies using nontransformed human cells demonstrated that IAA exposure modulated expression of genes that are involved in DNA DSB repair.15,50 The general pattern of cellular toxicity, genotoxicity and teratogenicity for the monoHAAs is IAA > bromocaetic acid (BAA) ≫ chloroacetic acid (CAA), and this pattern is repeated among bioassays in bacteria, mammalian cells, human cells, and mouse embryos.9,14,51 These toxicological end points are highly correlated with the dissociation energy of the halogen bonded to the α-carbon of the monoHAAs and to their relative SN2 reactivity. Thus, the physiochemical characteristics of the monoHAAs are directly related to their toxicity.14 Recently, it was reported that DNA was not the direct target of the monoHAAs, and CAA and IAA inhibited glyceraldehyde-3phosphodehydrogenase (GAPDH) without affecting other glycolytic enzymes.52,53 The quantitative toxicity of the monoHAAs and the inhibition kinetics of GAPDH also expressed the pattern of IAA > BAA ≫ CAA.51 The antioxidant butylated hydroxyanasol was effective in reducing the cytotoxicity and genotoxicity of IAA, and this result implies that IAAmediated reactive oxygen species generation plays a role in its toxicity.54 The genotoxicity tests indicated that IAA and IF might be potential genotoxic carcinogens. We used an in vitro cell transformation assay to determine if IAA or IF had carcinogenic potential.55 The biological end points of cell transformation included loss of contact inhibition, concanavlin A agglutination,

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-21-54237203. Fax: 86-21-64045165. E-mail: wdqu@ fudan.edu.cn. Author Contributions ∥

X.W., S.W., and W.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from the National Science Foundation (No. 30972438), National Key Technology R&D Program in the 11th Five-Year Plan (No. 2006BAI19B02), National High-Technology R&D Program (No. 2008AA062501-2 and 2013AA065204), Shanghai Municipal Health Bureau Leading Academic Discipline Project (No. 08GWD14), Nonprofit Foundation of National Health Ministry in the 12th Five-Year Plan (201002001, 201302004, and 2012BAJ25B05), and Dawn Scholarship Project (No. 07SG01). We thank Dong Zhang for collecting water samples and are grateful to Dr. Tao Jiang and Professor Nong Zhang (Department of Pathology, Fudan University) for their technical support in the histological analyses. We thank Dr. Michael J. Plewa for his crucial comments and suggestions (Global Safe Water Institute, University of Illinois at Urbana−Champaign) and Dr. William K. Kaufmann (Department of Pathology, University of North Carolina) for his manuscript editing. The authors would like to thank the four anonymous reviewers and the editor for their comments and suggestions.

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ABBREVIATIONS: Cyto B, cytochalasin B; CBMN, cytokinesis-block micronucleus; DBPs, disinfection byproducts; DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethyl sulfoxide; DSBs, doublestranded breaks; ECD, electron capture detection; FBS, fetal bovine serum; GC, gas chromatography; HAAs, haloacetic acids; H2O2, hydrogen peroxide; IAA, iodoacetic acid; iodo-acids, iodoacetic acids; IARC, International Agency for Research on Cancer; I-DBPs, iodinated disinfection by products; IF, iodoF

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form; iodo-THMs, iodotrihalomethanes; MLD, median lethal dose; MMC, mitomycin C; MX, 3-chloro-4-(dichloromethyl)-5hydroxy-2-(5H)-furanone; PBS, phosphate-buffered saline; SCGE, single-cell gel electrophoresis; TF, transformation frequency; THMs, trihalomethanes; U.S. EPA, United States Environmental Protection Agency; WHO, World Health Organization; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

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