Effect of Dibromoacetic Acid on DNA Methylation, Glycogen Accumulation, and Peroxisome Proliferation in Mouse and Rat Liver

Lianhui Tao1, Wei Wang, Long Li, Paula M. Kramer and Michael A. Pereira

Department of Pathology, Medical College of Ohio, Toledo, Ohio 43614-5806

Received May 18, 2004; accepted August 4, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dibromoacetic acid (DBA) is a drinking water disinfection by-product. Its analogs, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are liver carcinogens in rodents. We evaluated the ability of DBA to cause DNA hypomethylation, glycogen accumulation, and peroxisome proliferation that are activities previously reported for the two other haloacetic acids. Female B6C3F1 mice and male Fischer 344 rats were administered 0, 1,000, and 2,000 mg/l DBA in drinking water. The animals were euthanized after 2, 4, 7, and 28 days of exposure. Dibromoacetic acid caused a dose-dependent and time-dependent decrease of 20%–46% in the 5-methylcytosine content of DNA. Hypomethylation of the c-myc gene was observed in mice after 7 days of DBA exposure. Methylation of 24 CpG sites in the insulin-like growth factor 2 (IGF-II) gene was reduced from 80.2% ± 9.2% to 18.8% ± 12.9% by 2,000 mg/l DBA for 28 days. mRNA expression of the c-myc and IGF-II genes in mouse liver was increased by DBA. A dose-dependent increase in the mRNA expression of the c-myc gene was also observed in rats. In both mice and rats, DBA caused dose-dependent accumulation of glycogen and an increase of peroxisomal lauroyl-CoA oxidase activity. Hence, DBA, like DCA and TCA, induced hypomethylation of DNA and of the c-myc and IGF-II genes, increased mRNA expression of both genes, and caused peroxisome proliferation. Again like DCA, DBA also induced glycogen accumulation. These results indicate that DBA shares biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be a liver carcinogen.

Key Words: dibromoacetic acid; DNA hypomethylation; insulin-like growth factor 2; liver; c-myc; peroxisome proliferation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Haloacetic acids including dichloroacetic acid (DCA), trichloroacetic acid (TCA), and dibromoacetic acid (DBA) are found in finished drinking water, being formed as disinfection by-products (Arora et al., 1997Go; EPA, 1998; Boorman, 1999Go). The total concentration of haloacetic acids in drinking water was reported to be from 10 to 200 µg/l, with the concentration of DBA reported to be as high as 20 µg/l, with average and 90th percentile concentrations of 1.4 and 2.16 µg/l, respectively. Furthermore, when there is a significant quantity of bromide in the water, brominated haloacetic acids, especially DBA, can become the predominant form of haloacetic acid present in chlorinated drinking water. The widespread potential for human exposure to haloacetic acids in finished drinking water has raised questions about their health hazards. Concern over the health effects of chronic exposure to haloacetic acids on large populations led to a bioassay that demonstrated that DCA and TCA caused liver tumors in mice (Herren-Freund et al., 1987Go). Dichloroacetic acid has also been reported to cause liver tumors in rats (DeAngelo et al., 1996Go). The concentrations in drinking water of DCA and TCA that were used to demonstrate their carcinogenic activity in rodents was approximately 10,000-fold that found in drinking water. This identification of a potential carcinogenic hazard has led the US Environmental Protection Agency (USEPA) to classify DCA and TCA as probable and possible human carcinogens, respectively (EPA, 2003aGo, 2003bGo) and the International Agency for Research on Cancer to conclude that there is limited evidence in experimental animals for the carcinogenicity of the two chloroacetic acids (IARC, 1995Go).

Because much fewer toxicology and carcinogenicity data are available for DBA (Boorman, 1999Go), its long-term carcinogenicity is now being evaluated by the US National Toxicology Program. The carcinogenic activity of DCA and TCA appears not to result from genotoxic activity, as in vitro and in vivo tests for their genotoxicity have not demonstrated significant activity (Anna et al., 1994Go; Fahrig et al., 1995Go; Ferreira-Gonzalez et al., 1995Go; Fox et al., 1996Go; Meier and Blazak, 1990Go; Schroeder et al., 1997Go). The mechanism of DCA and TCA carcinogenicity has been hypothesized to be epigenetic, involving DNA methylation (Pereira et al., 2004aGo; Tao et al., 2000aGo, 2000bGo). DNA methylation as 5-methylcytosine (MeC) is an epigenetic mechanism that regulates chromosomal stability, histone acetylation, and the expression of genes (Eden et al., 2003Go; Feinberg and Tycko, 2004Go; Momparler, 2003Go; Razin and Shemer, 1999Go). DNA hypomethylation is a common early event in human and animal tumors that has been proposed to be a key factor in expanding the population of cells at risk for developing cancer (Baylin et al., 1998Go; Dunn, 2003Go). In mouse liver and hepatic tumors, we have reported that DCA and TCA caused global hypomethylation of DNA, as well as hypomethylation in the promoter regions of the insulin-like growth factor 2 (IGF-II), c-jun, and c-myc protooncogenes (Tao et al., 1998Go, 2000aGo, 2000bGo, 2004Go). We choose to evaluate the methylation of two of these genes, IGF-II and c-myc, because the expression of their mRNA is increased in mouse liver tumors. Other nongenotoxic mouse liver carcinogens, including trichloroethylene, 2,4-dichlorophenoxyacetic acid, dibutyl phthalate, gemfibrozil, Wyeth-14,643, and phenobarbital, have been shown to decrease the methylation of DNA (Counts et al., 1996Go, 1997Go; Ge et al., 2001Go, 2002Go; Tao et al., 1999Go). Thus, the ability of many nongenotoxic carcinogens to cause DNA hypomethylation suggests that it might be a common mechanism for their carcinogenic activity and could be useful as a biomarker to identify other nongenotoxic liver carcinogens.

Although DCA and TCA are structurally similar, differing by only one chlorine atom, their biochemical toxicology and histopathology have been shown to be quite different (Bull et al., 1990Go; Latendresse and Pereira, 1997Go; Pereira, 1996Go; Pereira and Phelps, 1996Go; Pereira et al., 1997Go, 2001Go; Tao et al., 1996Go;). For example, in female B6C3F1 mice, liver tumors induced by DCA were predominantly eosinophilic and contained glutathione S-transferase-{pi} (GST-{pi}), whereas those induced by TCA were basophilic and lacked GST-{pi} (Latendresse and Pereira, 1997Go; Pereira, 1996Go). Dichloroacetic acid–treated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes, while TCA caused a smaller increase in liver size without significant cytomegaly and glycogen accumulation (Bull et al., 1990Go). Both DCA and TCA are peroxisome proliferators; however, prolonged exposure to TCA, but not to DCA, resulted in maintenance of the increased activity of the peroxisomal enzyme, acyl-CoA oxidase (DeAngelo et al., 1989Go; Goldsworthy and Popp, 1987Go; Parrish et al., 1996Go). In the present study, we evaluated in the liver of female B6C3F1 mice and male F-344 rats the ability of DBA to cause (1) hypomethylation of DNA and the IGF-II and c-myc genes, (2) accumulation of glycogen, and (3) enhancement of peroxisome proliferation. Our goal was to determine whether the molecular and biochemical alterations induced by DCA and TCA were shared by DBA.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment. The exposure of animals was performed at Battelle Memorial Institute (Columbus, OH) under contracts from the National Institute of Environmental Health Sciences. Male Fischer 344 rats and female B6C3F1 mice at 24 to 30 days of age were obtained from Taconic Laboratory Animals and Service (Germantown, NY). The animals were group housed in solid polycarbonate cages in animal rooms maintained at 72° ± 3°F and 50% ± 15% relative humidity. The feed was NTP-2000 in meal form (Zeigler Brothers, Inc., Gardner, PA), and the drinking water was from the City of Columbus municipal supply. Both feed and water were supplied ad libitum. At 7–8 weeks of age, the animals were administered DBA at concentrations of 0, 1000, or 2000 mg/l in tap water, neutralized with sodium hydroxide to pH 5.0 to 7.0. The animals were euthanized after 2, 4, 7, and 28 days of exposure to DBA. At necropsy, the livers were collected, rapidly frozen in liquid nitrogen, and stored at –70°C.

Determination of the methylation of DNA and of the c-myc and IGF-II Genes. DNA was isolated from the liver by digestion with 400 µg/ml RNase A and 200 µg/ml proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol. Global methylation of DNA was determined by dot-blot analysis using a monoclonal antibody specifically against 5-methylcytosine, as described previously (Tao et al., 2004Go). DNA was dotted onto a Hybond nitrocellulose membrane. The membrane was then probed with a 1:1,000 dilution of mouse monoclonal primary antibody specific against 5-MeC (Eurogentec Company, Belgium), washed with T-BST (Tris-buffered saline plus Tween 20, pH7.6) and incubated with 1:2,000 dilution of horseradish peroxidase (HRP)–conjugated secondary anti-mouse-IgG antibody for 1 h. The membrane was then treated with enhanced-chemiluminescence Western blotting detection reagents and exposed to Kodak autoradiograph films. The optical density (OD) for the 5-MeC in DNA was measured with the Scion Image Analysis System (Scion Corp., Frederick, MD). Equal loading of the DNA was indicated by equal intensity of 0.02% methylene blue staining.

Methylation in the promoter region of the mouse c-myc gene was determined by digestion with the methylation-sensitive restriction endonuclease Hpa II followed by Southern blot analysis (Tao et al., 2000aGo 2000bGo). The digested DNA was electrophoresed on 1% agarose gel and transferred by vacuum to Hybond-N+ nylon membranes. The membranes were hybridized with a random 32P-labeled c-myc probe. Autoradiography was performed with Kodak Biomax MR X-ray film.

Methylation of CpG sites between exons 4 and 5 in the differentially methylated region-2 (DMR-2) of the mouse IGF-II gene was determined by a bisulfite-modified DNA sequencing procedure (Tao et al., 2004Go). The DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil, while methylated cytosine was resistant. Polymerase chain reaction amplification was performed with the following primers: upstream, 5'-TTG ATG GTA TTA TAT TGT AGA ATT AT-3' and downstream, 5'-AAC TAA AAT TAT CTA TCC TAT AAA AC-3' (Hemberger et al., 1998Go; Olek et al., 1996Go; Tao et al., 2004Go). The PCR-generated DNA fragments (616 bp) were ligated into the TA cloning vector, pCR 2.1 and transformed into One Shot TOP10F' Chemically Competent Escherichia coli cells according to standard protocols (Invitrogen, Carlsbad, CA). Plasmid DNA was automatically sequenced with an ABI Model 310 DNA sequencer (PerkinElmer, Norwalk, CT).

RT-PCR analysis for the mRNA expression of the c-myc and IGF-II genes. mRNA expression of the c-myc and IGF-II genes was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) as described previously (Tao et al., 2004Go). Total RNA was extracted from liver tissues using TRIzol Reagent and treated with DNase I. cDNA was synthesized from 1 µg of total RNA using avian myeloblastosis virus reverse transcriptase. The c-myc and IGF-II genes were respectively co-amplified with glyceraldehyde 3-phosphate dehydrogenase (Gapdh) used as an internal standard. Primer sequences for the c-myc gene (GenBank Database Accession Number: Z38066) were upstream: 5'-CTA CTC GCC TCC CTG CGA AG-3' (nt 318–337 bp) and downstream: 5'-GCG TAG CAG GCT CCA CGT CG-3' (nt 588–607 bp); for the IGF-II gene (GenBank Database Accession Number: NM_010514) were upstream: 5'-GGC CCC GGA GAG ACT CTG TGC-3' (nt 1014–1034 bp) and downstream: 5'-GAA GTC GTC CGG AAG TAC GG-3' (nt 1231–1250 bp); and for the Gapdh gene (GenBank Database Accession Number: NM_008084) were upstream: 5'-ATG GTG AAG GTC GGT GTG AAC G-3' (nt 47–68 bp) and downstream: 5'-GTT GTC ATG GAT GAC CTT GGC C-3' (nt 520–541 bp). The PCR products were electrophoresed and the optical density of the products was measured with the Scion Image Analysis System.

Determination of liver glycogen. Glycogen was isolated from 30–50 mg of liver by dissolving the tissue in 30% potassium hydroxide saturated with Na2SO4 for 30 min at 100°C, followed by ethanol precipitation. Glycogen content was determined by the phenol-sulfuric acid spectrophotometric method, as described previously (Pereira et al., 2004aGo) and expressed as micrograms of glycogen per milligram of liver.

Determination of peroxisomal lauroyl-CoA oxidase activity. Peroxisomal acyl-CoA oxidase activity was determined using lauroyl-CoA as the substrate and the fluorometric method based on hydrogen peroxide oxidation of 4-hydroxyphenylacetic acid (Kvannes and Flatmark, 1991Go; Poosch and Yamazaki, 1986Go). Liver was homogenized in 60 mM potassium phosphate (pH 7.0) containing 0.2% Triton X-100 and centrifuged at 1,000 x g. The 1,000 x g supernatant was diluted to 40 mg protein/ml and a 0.5 ml aliquot was added to 0.5 ml of a reaction mixture containing 0.2% Triton X-100, 60 mM potassium phosphate (pH 7.0), 1 mM 4-hydroxyphenylacetic acid, 4 U/ml HRP, 20 mM FAD, and 60 mM lauroyl-CoA. The reaction was incubated at 37°C for 30 min in a shaking water bath and then stopped by the addition of 2 mM KCN in 100 mM sodium bicarbonate, pH 10.5. Fluorescence was determined using excitation at 320 nm and emission at 405 nm. Peroxisomal lauroyl-CoA oxidase activity was expressed as nanomoles of hydrogen peroxide produced/min/mg protein.

Statistical analysis. The results were analyzed for statistical significance by one-way analysis of variance followed by the Bonferroni t-test. Statistical significance was indicated by a p value < 0.05 unless otherwise indicated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of DBA on DNA Methylation
Dibromoacetic acid administered in the drinking water caused DNA hypomethylation in the liver of both mice and rats (Fig. 1). Reduction of DNA methylation was significant after 7 days of exposure to DBA and remained suppressed for 28 days. Dibromoacetic acid exposure of shorter duration for 2 and 4 days did not significantly decrease DNA methylation. The reduction of DNA methylation was dose dependent, being reduced by 45% and 70% in mice and 33% and 52% in rats after 28 days of exposure to 1,000 and 2,000 mg/l DBA, respectively. Hence, DBA caused a time-dependent and dose-dependent hypomethylation of hepatic DNA in mice and rats.



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FIG. 1. Effect of DBA on DNA methylation in the livers of female B6C3F1 mice and male Fischer 344 rats. Mice (A) and rats (B) were administered DBA at concentrations 0, 1000, 2000 mg/l in their drinking water and were euthanized 0, 2, 4, 7, and 28 days later. The results are means ± S.E. from eight animals/group. The asterisk indicates statistical difference from the untreated control group, p value < 0.05.

 
Effect of DBA on the Methylation of the c-Myc and IGF-II Genes
Southern blots of Hpa II-digested DNA from the liver of mice exposed to 2,000 mg/l of DBA for 7 days contained bands of 0.2, 0.4, 0.5, 1.0, and 2.2 kb when probed for the c-myc promoter region (Fig. 2). These bands were absent on the blots when the DNA was isolated from mice exposed for 0, 2, or 4 days to DBA and the DNA was not digested with Hpa II (data not shown). The probed region of the c-myc gene contains 12 CCGG sites, each of which would be resistant to cleavage by Hpa II when the internal cytosine is methylated. Thus, the internal cytosine of these CCGG sites would appear to be methylated, preventing digestion by Hpa II; yet after 7 days of exposure to DBA the internal cytosine of some of the CCGG sites became demethylated, thereby allowing digestion by Hpa II.



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FIG. 2. Effect of DBA on the methylation of the c-myc gene in the livers of female B6C3F1 mice. Mice were administered DBA at concentrations 0 and 2,000 mg/l in their drinking water and were euthanized 0, 2, 4, and 7 days later. DNA was extracted from mouse liver and digested with methylation-sensitive restriction endonuclease Hpa II, followed by Southern blot analysis probing for the c-myc gene (A). The arrows in the right margin indicate the size of the restriction fragments. Equal loading of DNA in the lanes of the gel was indicated by equal ethidium bromide fluorescence (B).

 
The bisulfite-modified DNA sequencing procedure was employed to determine the methylation status of 24 CpG sites in the DMR-2 region between exons 4 and 5 of the mouse IGF-2 gene. The sequence of this region obtained after bisulfite-treatment of the samples used in this study was consistent with what we have previously published (Tao et al., 2004Go). The methylation status of the 24 CpG sites in the probed DMR-2 region is presented in Figure 3. The number of CpG sites that were methylated in the liver from control mice ranged from 16–21, whereas after exposure to 2,000 mg/l DBA for 28 days only 0–7 sites were methylated. Thus, the percentage of the CpG sites that were methylated was reduced from 80.2% ± 4.6% (mean ± S.E.) in control mice to 18.7% ± 6.4% in mice exposed to DBA (p < 0.001). The reduction in methylation at the CpG sites occurred mainly between sites 191 and 505 in the DMR-2 region.



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FIG. 3. Effect of DBA on CpG methylation in the DMR-2 of the mouse IGF-II gene. Female B6C3F1 mice were exposed to 0 and 2,000 mg/l DBA for 28 days. The numbers at the top of the accompanying table identify the location of the cytosine at the CpG sites. A bold capitalized letter M in the table indicates that the cytosine was methylated, whereas a capitalized letter U indicates that it was not methylated. The first column contains the identification number of the mice from which the sample was obtained.

 
mRNA Expression of the c-myc and IGF-II Genes
The effect of DBA on the mRNA level of the c-myc gene is presented in Figure 4. The ratio of the optical density of the RT-PCR product for c-myc to the product for Gapdh (housekeeping gene) is presented in Figures 4B and 4D. In mice, the expression of mRNA of the c-myc gene was increased 1.5–fold and 4.1–fold by 1,000 mg/l DBA and 2.5–fold and 5.1–fold by 2,000 mg/l DBA after exposure for 7 and 28 days, respectively (p value < 0.01; Figs. 4A and 4B). In rats, the expression of mRNA of the c-myc gene was increased 1.5–fold and 5.1–fold by 1,000 mg/l DBA and 3.8–fold and 5.8–fold by 2,000 mg/l DBA after exposure for 7 and 28 days, respectively (p value < 0.01; Figs. 4C and 4D). Thus, DBA caused a time-dependent and dose-dependent increase in the mRNA expression of the c-myc gene in mice and rats.



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FIG. 4. Effect of DBA on the mRNA expression of the c-myc gene in female B6C3F1 mice and male Fischer 344 rats. The animals were administered 0, 1,000, 2,000 mg/l DBA for 0, 2, 4, 7, and 28 days and then euthanized. Total liver RNA was extracted, and c-myc and Gapdh genes were co-amplified by RT-PCR. Panels A and C are the photographs of gels that represent the samples from mice and rats, respectively. The first lane of each gel contains HindIII restriction fragments of lambda-phage DNA used as molecular size markers. Panels B and D are optical density of the RT-PCR product of the c-myc gene divided by the density of the product for the Gapdh gene. Results are expressed as means ± S.E. for eight animals. The asterisk indicates statistical difference from the corresponding untreated control group (p value < 0.05).

 
The effect of DBA on the mRNA level of the IGF-II gene is presented in Figure 5. The ratio of the optical density of the RT-PCR product for IGF-II to the product for Gapdh (housekeeping gene) is presented in Figure 5B. The expression of mRNA of the IGF-II gene was increased 2.1–fold and 3.5–fold by 2,000 mg/l DBA after exposure for 7 and 28 days, respectively (p value < 0.01).



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FIG. 5. Effect of DBA on the mRNA expression of the IGF-II gene in female B6C3F1 mice. The animals were administered 0 and 2,000 mg/l DBA for 0, 4, 7, and 28 days and then euthanized. Total liver RNA was extracted, and IGF-II and Gapdh genes were co-amplified by RT-PCR. Panel A is a representative photograph of the gel under ultraviolet irradiation. The first lane of the gel contains HindIII restriction fragments of lambda-phage DNA used as molecular size markers. Panel B is the optical density of the RT-PCR product of the IGF-II gene divided by the density of the product for the Gapdh gene. Results are expressed as means ± S.E. for eight animals. The asterisk indicates statistical difference from the corresponding untreated control group (p value < 0.05).

 
Liver Glycogen Accumulation
Dibromoacetic acid treatment induced glycogen accumulation in the liver of mice and rats (Table 1). In mice, hepatic glycogen content was significantly increased from 60.4 ± 3.1 (mean ± S.E.) to 71.4 ± 2.9 mg/g liver or by 118% (p value < 0.01) after only 2 days of exposure to the high concentration of DBA (2,000 mg/l) and remained elevated for 28 days. The low concentration of DBA also increased liver glycogen in mice. In rats, exposure for 2 days to the high concentration of DBA was not sufficient to increase hepatic glycogen level, whereas exposure for 4 days did increase glycogen levels from 47.7 ± 1.1 to 58.8 ± 2.4 mg/g liver, or by 123% (p value < 0.01). The low concentration of DBA did not result in a statistically significant increase in liver glycogen until 28 days of exposure. Hence, DBA increased liver glycogen in both mice and rats, although a longer duration of exposure appeared to be required in rats.


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TABLE 1 Effect of DBA on Glycogen Accumulation in Mouse and Rat Liver

 
Peroxisome Proliferation
Dibromoacetic acid caused hepatic peroxisome proliferation as measured by lauroyl-CoA oxidase activity in both mice and rats (Table 2). A significant increase in lauroyl-CoA oxidase activity in the liver was observed in mice after exposure to 1,000 mg/l DBA for 7 days and 2,000 mg/l for 4 days (p values < 0.01); the high concentration of DBA caused a greater increase in lauroyl-CoA oxidase activity. In rats, lauroyl-CoA oxidase activity in the liver was significantly increased only by the high concentration of DBA after 2 days of exposure and remained elevated for 28 days (p values < 0.01).


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TABLE 2 Effect of DBA on Peroxisomal Lauroyl-CoA Oxidase Activity in Mice and Rats

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dibromoacetic acid is found in drinking water as a by-product of chlorine disinfection but data are limited concerning its health effects including carcinogenicity. However, two other haloacetic acids found in drinking water, DCA and TCA, have been shown to be hepatocarcinogens in mice and DCA is a hepatocarcinogen in rats (DeAngelo et al., 1996Go). We have previously shown that mouse liver tumors promoted by DCA or TCA exhibited global DNA hypomethylation and decreased methylation of the c-jun, c-myc, and IGF-II protooncogenes (Tao et al., 1998Go, 2000bGo, 2004Go). Furthermore, short-term (days) treatment with either DCA or TCA induced hypomethylation of DNA and of the c-jun, c-myc, and IGF-II protooncogenes in mouse liver (Pereira et al., 2001Go; Tao et al., 1998Go, 2000aGo, 2004Go). Other nongenotoxic liver carcinogens such as trichloroethylene, trihalomethanes (including chloroform), peroxisome proliferators, and phenobarbital have also been shown to induce hypomethylation of DNA and protooncogenes in mouse liver (Counts et al., 1996Go; Coffin et al., 2000Go; Ge et al., 2001Go and 2002Go; Pereira et al., 2001Go; Tao et al., 1999Go). Nongenotoxic carcinogens also induce DNA hypomethylation in other target organs, e.g., bromodichloromethane, bile acid, and rutin in rat colon (Pereira, et al., 2004bGo).

DNA hypomethylation has been shown to release methyl binding proteins and associated histone deacetylase that increases the acetylation of histones and opens up the chromatin to allow the binding of transcription factors and RNA polymerase to the promoter sequences of the target genes, resulting in increased expression of the genes (Dunn, 2003Go; Eden et al., 2003Go; Feinberg and Tycko, 2004Go; Momparler, 2003Go). DNA hypomethylation has also been proposed to promote the hypermethylation of tumor suppressor genes resulting in their downregulation (Dunn, 2003Go). The increased expression of protooncogenes and the downregulation of tumor suppressor genes are expected to result in a growth advantage for the affected cells that enhances the risk of cancer. This has led to DNA hypomethylation being proposed as a factor in the carcinogenic mechanism of nongenotoxic liver carcinogens (Counts and Goodman, 1995Go; Pereira et al., 2004aGo, 2004bGo; Tao et al., 2000aGo, 2004Go). It was further proposed that the ability to induce DNA hypomethylation could be used to screen chemicals for nongenotoxic carcinogenic activity.

Dibromoacetic acid induced hypomethylation of DNA, and of the promoter regions of the c-myc and IGF-II genes. DBA also increased the mRNA expression of the two genes that was associated with their hypomethylation. Dichloroacetic acid and TCA also induced hypomethylation of DNA and of the two genes, and increased the expression of the genes (Tao et al., 1998Go, 2000aGo, 2000bGo, 2004a). When administered at concentrations similar to those of the DBA used in our study, DCA and TCA have been shown to be mouse liver carcinogens (Bull et al., 1990Go; Herren-Freund et al., 1987Go; Pereira, 1996Go; Pereira and Phelps, 1996Go). Hence, using the concentrations of DCA and TCA that were high relative to their concentration found in drinking water, these studies identified a potential carcinogenic hazard for the two chloroacetic acids (IARC, 1995Go). The ability of DBA to induce hypomethylation of DNA, c-myc, and IGF-II would suggest that it is also a nongenotoxic liver carcinogen in mice, albeit at the concentrations higher than those found in drinking water.

The two haloacetic acids, DCA and TCA, appeared to elicit different histopathology (Bull et al., 1990Go; Herren-Freund et al., 1987Go; Latendresse and Pereira, 1997Go; Pereira, 1996Go; Pereira and Phelps, 1996Go). Dichloroacetic acid, but not TCA, induces glycogen accumulation in mouse liver. Dibromoacetic acid also induces glycogen accumulation in both mouse and rat liver, suggesting that its activity might be more similar to DCA than to TCA. Both DCA and TCA induce peroxisome proliferation in rodent liver (DeAngelo et al., 1989Go; Goldsworthy and Popp, 1987Go; Parrish et al., 1996Go), and we report here that DBA also induces peroxisome proliferation in mouse and rat liver. Liver tumors induced by TCA have been demonstrated to be basophilic staining, similar to those induced and promoted by other peroxisome proliferators. In contrast, tumors induced by DCA, especially in female mice, have been demonstrated to be eosinophilic staining. Should DBA prove to be a liver carcinogen, the associated DNA hypomethylation as well as the associated increased expression of protooncogenes, glycogen accumulation, and peroxisome proliferation could be involved in the carcinogenic process. Furthermore, these associated biochemical and molecular characteristics could be used to distinguish whether the development and histopathology of the tumors are more similar to those induced by DCA or TCA.


    ACKNOWLEDGMENTS
 
This research was supported in part by grant R82808301 from the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) program and grant 1 R03 ES10537–01 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA.


    NOTES
 

1 To whom correspondence should be addressed at Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine and Public Health, The Ohio State University, 300 West Tenth Avenue, 1148 CHRI, Columbus, OH 43210; Fax: (614)-293-4072. E-mail: tao-2{at}medctr.osu.edu.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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