Abstract Dichloroacetic acid (DCA) is a hepatocellular carcinogen in the male B6C3F1 mouse. Histopathologic analysis of a timed DCA dose response study has demonstrated three hepatic lesion sequences in this model. The purpose of the present study of 465 B6C3F1 male mice was to determine: (1) the persistence of early lesions in a “stop” study and (2) the efficacy of phenobarbital (PB) to promote these early lesions. The persistence of early lesions in mice exposed to drinking water containing 3.5 g/l DCA for 10 week intervals to 50 weeks was determined. One group remained exposed until the study was terminated at 100 weeks. Control animals received distilled water. In the promotion study, groups of animals exposed to drinking water containing 3.5 g/l DCA for 30 weeks were transferred to 0.6% phenobarbital (PB) or water alone and terminated at 60 or 90 weeks. Histopathologic analysis of liver lesions was performed on 1,481 formalin fixed, paraffin embedded tissue sections stained with hematoxylin and eosin. Lesions were identified and classified as altered foci (AF), hyperplastic nodules (HN), adenomas (AD) or carcinomas (CA). 10-50 weeks of DCA exposure doubled the prevalence of atypical (irregular misshapen and hyperchromatic) nuclei (82-95% vs 32% in untreated animals). Lifetime exposure to DCA was not required for induction of neoplastic lesions. Numerous neoplasms arose in animals exposed to 3.5 g/L DCA for 10, 20, 30, 40, or 50 weeks and then placed on water until 100 weeks. The prevalence of AD and CA increased with time to 50 weeks but decreased after 100 weeks, possibly reflecting the dose dependent suppression of proliferation in premalignant hepatic lesions such as hyperplastic nodules and adenomas by DCA. DCA exposure for 10-30 weeks doubled the prevalence of enlarged nuclei which were highly correlated with zonal changes. With increasing DCA exposure there was a shift in frequency of lesion types from hyperplastic nodues (20 weeks) to adenomas (40 weeks) to carcinomas (50 weeks). The major effect of PB promotion was to increase the number of eosinophilic AD at 60 and 90 weeks. These data demonstrate the continued development of preneoplastic hepatocellular lesions following termination of DCA treatment and are in agreement with studies which demonstrated that early-life exposure was as carcinogenic as life-long exposures for 98 weeks. Histopathologic analysis of these (DCA) stop and promotion studies in the B6C3F1 male mouse confirms that carcinomas can arise directly from single initiated cells in liver as well as within hyperplastic nodules and adenomas. Potentially, epigenetic-mediated effects may be involved since earlier reports indicate that RNA and micro-RNA profiles associated with prior DCA treatment were not sustainable. This work as supported by USEPA Cooperative Agreement #CR-814803-01-0. Citation Format: Julia H. Carter, Harry W. Carter, Anthony B. DeAngelo. Multiple hepatic lesion sequences in dichloroacetic acid (DCA) stop and promotion studies in the B6C3F1 male mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5049.

Early-life environmental factors can influence later-life susceptibility to cancer. Recent evidence suggests that metabolic pathways may mediate this type of latency effect. Previously, we reported that short-term exposure to dichloroacetic acid (DCA) increased liver cancer in mice 84 weeks after exposure was stopped. Here, we evaluated time course dynamics for key events related to this effect. This study followed a stop-exposure design in which 28-day-old male B6C3F1 mice were given the following treatments in drinking water for up to 93 weeks: deionized water (dH2O, control); 3.5 g/l DCA continuously; or 3.5 g/l DCA for 4-52 weeks followed by dH2O. Effects were evaluated at eight interim time points. A short-term biomarker study was used to evaluate DCA effects at 6, 15, and 30 days. Liver tumor incidence was higher in all DCA treatment groups, including carcinomas in 82% of mice previously treated with DCA for only 4 weeks. Direct effects of DCA in the short-term study included decreased liver cell proliferation and marked mRNA changes related to mitochondrial dysfunction and altered cell metabolism. However, all observed short-term effects of DCA were ultimately reversible, and prior DCA treatment did not affect liver cell proliferation, apoptosis, necrosis, or DNA sequence variants with age. Key intermediate events resulting from transient DCA exposure do not fit classical cytotoxic, mitogenic, or genotoxic modes of action for carcinogenesis, suggesting a distinct mechanism associated with early-life metabolic disruption.

This study demonstrates latent carcinogenic effects of the metabolic programming agent dichloroacetic acid following transient exposure in mice. Our findings highlight the potential for early-life postnatal changes in cell metabolism to alter cancer risk later in life.Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH(2)O, control); dH(2)O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH(2)O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (a parts per thousand yen85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.

As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV+chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.

More efficient models are needed to assess potential carcinogenicity hazard of environmental chemicals based on early events in tumorigenesis. Here, we investigated time course profiles for key events in an established cancer mode of action. Using a case study approach, we evaluated two reference phthalates, di(2-ethylhexyl) phthalate (DEHP) and its stereoisomer di-n-octyl phthalate (DNOP), across the span of a two-year carcinogenicity bioassay. Male B6C3F1 mice received diets with no phthalate added (control), DEHP at 0.12, 0.60, or 1.20%, or DNOP at 0.10, 0.50, or 1.00% (n = 80-83/group) for up to 104 weeks with six interim evaluations starting at week 4. Mean phthalate doses were 139, 845, and 3147 mg/kg/day for DEHP and 113, 755, and 1281 mg/kg/day for DNOP groups, respectively. Incidence and number of hepatocellular tumors (adenoma and/or carcinoma) were greater at ≥ 60 weeks for all DEHP groups with time and dose trends, whereas DNOP had no significant effects. Key events supported a peroxisome proliferator-activated receptor alpha (PPARα) mode of action for DEHP, with secondary cytotoxicity at the high dose, whereas DNOP induced modest increases in PPARα activity without proliferative or cytotoxic effects. Threshold estimates for later tumorigenic effects were identified at week 4 for relative liver weight (+24%) and PPARα activity (+79%) relative to the control group. Benchmark doses (BMDs) for these measures at week 4 clearly distinguished DEHP and DNOP and showed strong concordance with values at later time points and tumorigenic BMDs. Other target sites included testis and kidney, which showed degenerative changes at higher doses of DEHP but not DNOP. Our results highlight marked differences in the chronic toxicity profiles of structurally similar phthalates and demonstrate quantitative relationships between early bioindicators and later tumor outcomes.

Abstract Epidemiological studies have linked the consumption of disinfected surface waters to an increased risk of colorectal cancer. Approximately 600 disinfection byproducts (DBPs) have been identified for a number of disinfectants currently in use. An in-depth mechanism-based structure activity analysis undertaken identified 50 unregulated DBPs with the highest risk carcinogenic potential. Because it would be financially prohibitive to test all priority DBPs in chronic animal bioassays, we are developing an in vitro model system to identify DBPs of carcinogenic potential in normal human colonocytes. Specifically, a mixed culture of human colon mucosal cells including stem cells with the capability for differentiation (NCM460) were treated with brominated DBPs bromochloroacetic acid (BCAA), dibromonitromethane (DBNM), and tribromonitromethane (TBNM), as well as a positive colon carcinogenic control, azoxymethane. To measure cancer-related pathways being activated, global gene expression analysis of 10-day 10−6M treated NCM460 monolayers was performed on Affymetrix Human Genome U133 Plus 2.0 arrays and statistically significant genes were identified using one-way ANOVA with a false discovery rate (Benjamini-Hochberg test) of ≤ 0.05 followed by a post-hoc test (Scheffe). Pathway analysis was performed using DAVID bioinformatics resource (National Institutes of Health) which identified multiple pertinent pathways activated with all or a majority of the treatments including cell adherins, ubiquitin-mediated proteolysis and xenobiotic metabolism. Of significance, AOM, DBNM and TBNM treatments induced genes involved in the WNT/β-catenin, a well studied signaling pathway that modulates cell proliferation and differentiation and is a hallmark pathway for cancer development. As validation, immunocytochemical staining for β-catenin indicated increased nuclear translocation after treatment with all DBPs and AOM when compared to the control. In addition, real-time RT-PCR analysis measured expression of select genes known to be directly regulated by β-catenin after 7 and 10-day 10−6M AOM and TBNM treatment. MYC, AXIN2 and PPARD expression significantly increased (p<0.05, t-test) with 10, but not 7, day treatment with both chemicals, suggesting WNT/β-catenin signaling pathway activation at the later time point. Importantly, preliminary studies indicate colony transformation of these cells after DBP treatment as measured in soft agar assay. Our overall results suggest the tested water DBPs may activate cellular mechanisms that mediate colon cancer development and etiology. Additionally, we demonstrate this in vitro methodology can detect carcinogenic potential of chemicals and may serve as a useful tool to test other chemicals in a more cost-effective and expedited manner than traditional animal bioassays. [Abstract does not necessarily reflect the policies of the U.S. EPA.] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 744. doi:1538-7445.AM2012-744

Abstract The carcinogenic potential of chlorinated organics is of direct importance in human risk assessment. Most drinking water chlorinated organics are disinfection by products (DBPs) of water chlorination and many test positive in rodent bioassays. Trihalomethanes (THMs) are the most prevalent DBPs generated in chlorinated drinking water and chloroform (TCM) is the THM in highest concentration in finished drinking water. Human exposure to TCM occurs through ingestion of drinking water, inhalation and dermal exposure. TCM is carcinogenic to the liver and kidney of rodents including male and female B6C3F1 mice and male F-344/N and Osborne-Mendel rats. The carcinogenic mechanism of TCM in the rodent is not completely understood. Four mechanisms have been proposed: 1) mutagenicity; 2) reparative hyperplasia; 3) altered gene expression; and 4) secondary genotoxicity. Here, histopathology, immunohistochemistry, and quantitative image analysis were used to examine reparative cell proliferation and altered gene expression as potential mechanisms of TCM-induced hepatocellular carcinogenesis in male F-344/N rats. Animals were exposed to concentrations of 803 + 5 or 1592 + 21 mg/L in the drinking water for 78 or 100 weeks. Distilled water was the vehicle control. The high TCM dose increased the prevalence (% of animals with a lesion) of hepatocellular neoplasia (carcinoma and adenoma) 17.5% vs. 5.1% (p<0.05) and marginally enhanced the prevalence of combined preneoplastic and neoplastic tumors (20.5% vs 7.7%; 0.05 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3451.

BACKGROUND:Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC).RESULTS:To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag) expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-gamma and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-gamma metabolic responses were functional in both cell lines, but IFN-gamma anti-proliferative responses functioned only in tumor cells.CONCLUSIONS:Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

Bromate, a common disinfectant byproduct of drinking water ozonation, has been linked to human and animal renal toxicity, including renal cell carcinomas in multiple animal species. Here, we evaluate changes in protein and gene expression through two-dimensional difference gel electrophoresis (2D-DIGE) and Affymetrix arrays to identify potential modes of action involved in potassium bromate carcinogenicity. Male rats were exposed to potassium bromate in drinking water at concentrations of 0, 1, 20 and 400ppm for two weeks. Differential expression of glycolytic proteins including enolase 1 (Eno1), triosephosphate isomerase 1 (Tpi1) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) suggests that bromate toxicity is associated with changes in energy consumption and utilization in renal cells involving up-regulation of glycolytic processes that may be the result of altered mitochondrial function. Several alterations in glycolysis and mitochondrial gene transcripts were also observed to be consistent with this mode of action. These studies provide insight into early events in renal cell physiology altered by bromate exposure.

The prevalence (percent of animals with a tumor) and multiplicity (number of tumors per animal) of hepatocellular neoplasia in the male B6C3F1 mouse exposed to trichloroacetic acid (TCA) in the drinking water were determined. Male mice were exposed to 0.05, 0.5, and 5 g/L TCA for 60 wk (Study 1), to 4.5 g/L TCA for 104 wk (Study 2) and to 0.05 and 0.5 g/L TCA for 104 wk (Study 3). Time-weighted mean daily doses measured for the low, medium, and high dose groups were consistent over the three studies, 6-8, 58-68, and 572-602 mg/kg-d for the 0.05, 0.5, and the 4.5-5 g/L treatment groups, respectively. No significant changes in animal survival were noted across the studies. A significant increase in the prevalence and multiplicity of hepatocellular tumors was found in the 58-68 and 572-602 mg/kg/d TCA dose groups. Nonhepatoproliferative changes (cytoplasmic alterations, inflammation, and necrosis) in mice treated with TCA were mild and dose related. A TCA-induced increase in liver palmitoyl CoA oxidase activity, a marker of peroxisome proliferation, correlated with tumor induction. A linear association was found between peroxisome proliferation and tumor induction. Sporadic increases in the labeling index of nuclei outside of proliferative lesions were observed at carcinogenic doses throughout the studies. Given that there are no compelling data demonstrating genotoxic activity of either TCA or any metabolite, data are consistent with an epigenetic mode of action. The studies provide dose-response data on the development of hepatocellular neoplasia in male mice over a lifetime exposure to TCA. A no-observed-effect-level (NOEL) of 6 mg/kg/d was calculated for neoplastic and nonproliferative liver pathology.

Large-scale differential gene expression analysis was used to examine the biological effects of disinfected surface waters on cultured rat hepatocytes. Source water from East Fork Lake (Harsha Lake), a reservoir on the Little Miami River in Ohio, was spiked with iodide and bromide and disinfected by chlorination or ozonation/postchlorination. The chlorinated and ozonated/postchlorinated waters were concentrated, respectively, 136- and 124-fold (full strength) by reverse-osmosis membrane techniques. Volatile disinfection by-products (DBP) lost during concentration were restored to the extent possible. Primary rat hepatocytes were exposed to either full-strength or 1:10 or 1:20 dilutions of the concentrates for 24 h and assayed for cytotoxicity and gene expression alterations. The full-strength concentrates were cytotoxic, whereas the diluted samples exhibited no detectable cytotoxicity. Differential gene expression analysis provided evidence for the underlying causes of the severe cytotoxicity observed in rat hepatocytes treated with the full-strength ozonation/postchlorination concentrate (e.g., cell cycle arrest, metabolic stasis, oxidative stress). Many gene expression responses were shared among the hepatocyte cultures treated with dilutions of the ozonation/ postchlorination and chlorination concentrates. The shift in the character of the response between the full-strength concentrates and the diluted samples indicated a threshold for toxicity. A small subset of gene expression changes was identified that was observed in the response of hepatocytes to peroxisome proliferators, phthalate esters, and haloacetic acids, suggesting a peroxisome proliferative response.

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose-response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose-response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka (Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N(7)-methylguanine and O(6)-methylguanine in the liver; mutation frequency using lambda cII transgenic medaka and lambda cII transgenic (Big Blue(R)) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using "alternative" models.

BACKGROUND:Potassium bromate (KBrO3), used in both the food and cosmetics industry, and a drinking water disinfection by-product, is a nephrotoxic compound and rodent carcinogen. To gain insight into the carcinogenic mechanism of action and provide possible biomarkers of KBrO3 exposure, the gene expression in kidneys from chronically exposed male F344 rats was investigated.METHODS:Male F344 rats were exposed to KBrO3 in drinking water for 52 and 100 wk. Kidneys were removed, frozen, and stored at -80°C, then used for Affymetrix microarray analysis. Gene expression patterns were examined using a non-carcinogenic (20 ppm) and carcinogenic dose (400 ppm) at 52 wk, and compared to 100 wk high dose (400 ppm) and adenoma gene expression.RESULTS:Statistical analysis revealed 144, 224, 43, and 994 genes out of 15866 from the 52 wk low, 52 wk high, 100 wk high, and adenomas respectively, were differentially expressed when compared to control kidneys. Gene ontology classification of the 52 wk high dose showed alterations of gene transcripts involved in oxidative stress, lipid metabolism, kidney function/ion transport, and cellular function. In a comparison of kidney development gene expression, alterations were seen in the adenomas but not in the 52 wk bromate-treated kidneys. However, the normal kidney from the high dose group resembled the adenoma expression pattern with early kidney development genes being up-regulated and adult phase genes being down-regulated. Moreover, eight genes were identified which could serve as biomarkers of carcinogenic exposure to bromate. The most promising of these was Pendrin, or Slc26a4, a solute carrier of chloride and iodide active in the kidney, thyroid, and inner ear. All these tissues are targets of KBrO3 toxicity. Expression array results were verified with quantitative real-time rtPCR.CONCLUSIONS:These data demonstrate that the 400 ppm carcinogenic dose of KBrO3 showed marked gene expression differences from the 20 ppm non-carcinogenic dose. Comparison of kidney development gene expression showed that the adenoma patterns were more characteristic of embryonic than adult kidneys, and that the normal kidney from the high dose group resembled the adenoma-like gene expression pattern. Taken together, the analysis from this study identifies potential biomarkers of exposure and illuminates a possible carcinogenic mode of action for KBrO3.

BACKGROUND: Epidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water. METHODS: DNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB. RESULTS: CCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs. CONCLUSION: CCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

Chlorination is a widely used method for disinfection of drinking water supplies. Reaction of chlorine with naturally present organic compounds can result in toxic by-products. One major disinfection by-product from the chlorination of drinking water is dichloroacetic acid (DCA). This chemical has been shown to be carcinogenic in rodents, yet little genotoxicity data are available to assess the possible role of DNA and/or chromosomal damage in this process. We have used the peripheral blood erythrocyte micronucleus (MN) assay and the alkaline single cell gel electrophoresis (SCG) technique to investigate the in vivo genotoxicity of DCA in bone marrow and blood leukocytes, respectively. The MN assay detects chromosome breakage and/or malsegregation, while the SCG assay detects DNA damage (e.g., single strand breaks, alkali-labile sites, crosslinking). Mice were exposed to this compound in drinking water, available ad libitum, for up to 31 weeks. Our results show a small but statistically significant dose-related increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) after subchronic exposure to DCA for 9 days. In addition, at the highest dose of DCA tested (3.5 g/l), a small but significant increase in the frequency of micronucleated normochromatic erythrocytes (NCE) was detected following exposure for ≥ 10 weeks. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage. Based on the lack of any difference observed in the proportion of kinetochore-positive micronuclei between the treated and control animals, we interpret the induced MN as arising from clastogenic events. The SCG technique suggested the presence of DNA crosslinking in blood leukocytes in mice exposed to 3.5 g/l DCA for 28 days. These data provide evidence that DCA may be an extremely weak inducer of chromosome damage when provided to mice in drinking water under conditions which lead to increased levels of tumors. © 1996 Wiley-Liss, Inc.