DNA Hypomethylation Induced by Drinking Water Disinfection By-Products in Mouse and Rat Kidney
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《毒物学科学杂志》
Department of Internal Medicine, Division of Hematology and Oncology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210
Department of Pathology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, Ohio 43614
ABSTRACT
Bromodichloromethane (BDCM), chloroform, dibromoacetic acid (DBA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) are chlorine disinfection by-products (DBPs) found in drinking water that have indicated renal carcinogenic and/or tumor promoting activity. We have reported that the DBPs caused DNA hypomethylation in mouse liver, which correlated with their carcinogenic and tumor promoting activity. In this study, we determined their ability to cause renal DNA hypomethylation. B6C3F1 mice were administered DCA or TCA concurrently with/without chloroform in their drinking water for 7 days. In male, but not female mouse kidney, DCA, TCA, and to a lesser extent, chloroform decreased the methylation of DNA and the c-myc gene. Coadministering chloroform increased DCA but not TCA-induced DNA hypomethylation. DBA and BDCM caused renal DNA hypomethylation in both male B6C3F1 mice and Fischer 344 rats. We have reported that, in mouse liver, methionine prevented DCA- and TCA-induced hypomethylation of the c-myc gene. To determine whether it would also prevent hypomethylation in the kidneys, male mice were administered methionine in their diet concurrently with DCA or TCA in their drinking water. Methionine prevented both DCA- and TCA-induced hypomethylation of the c-myc gene. The ability of the DBPs to cause hypomethylation of DNA and of the c-myc gene correlated with their carcinogenic and tumor promoting activity in mouse and rat kidney, which should be taken into consideration as part of their risk assessment. That methionine prevents DCA- and TCA-induced hypomethylation of the c-myc gene would suggest it could prevent their carcinogenic activity in the kidney.
Key Words: dichloroacetic acid; trichloroacetic acid; DNA hypomethylation; kidney.
INTRODUCTION
Trihalomethanes including chloroform and bromodichloromethane (BDCM) and haloacetic acids including dichloroacetic acid (DCA), trichloroacetic acid (TCA), and dibromoacetic acid (DBA) are major organic disinfection by-products (DBPs) in finished drinking water after chlorination treatment (Chen and Weisel, 1998; Uden and Miller, 1983). The disinfection by-products, with the exception of DBA for which the results of a chronic bioassay has yet to be published, have demonstrated carcinogenic activities in laboratory rodents (U.S. EPA, 2004a–d; IARC, 1991, 2004). Chloroform, BDCM, DCA, and TCA have been demonstrated to be carcinogens and/or tumor promoters in mouse and rat liver (U.S. EPA, 2004a–d; IARC, 1991, 2004; NCI, 1976). The kidney has also been identified as a potential target organ for these disinfection by-products. Chloroform has been reported to induce renal tumors in male mice and rats (Jorgenson et al., 1985; NCI, 1976; Roe et al., 1979). BDCM significantly increased the yield of kidney tumors in male B6C3F1 mice and in both sexes of Fischer 344 rats when administered by gavage (IARC, 1991; NTP, 1987). In N-methyl-N-nitrosourea (MNU)-initiated male, but not female B6C3F1 mice, TCA promoted kidney tumors while DCA did so only when coadministered with chloroform in drinking water (Pereira et al., 2001). DBA has been shown to share numerous biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be carcinogenic in the kidney (Tao et al., 2004b).
Carcinogens are generally considered to increase the risk of cancer by two different mechanisms: genotoxic and epigenetic mechanisms. DNA methylation is a fundamental epigenetic process that not only modulates gene transcription, but is also key to histone acetylation and chromosomal stability. Global hypomethylation of DNA has been proposed to contribute to carcinogenesis (Baylin, 2002; Goodman and Watson, 2002). Hypomethylation of DNA and/or c-myc protooncogene has also been demonstrated in mouse liver and hepatic tumors in response to many nongenotoxic carcinogens, including chloroform, BDCM, DCA, TCA, peroxisome proliferators, and phenobarbital (Coffin et al., 2000; Counts et al., 1996; Ge et al., 2001, 2002; Tao et al., 1998, 2000a,b, 2004a,b). Furthermore, BDCM induces DNA hypomethylation in rat, but not mouse colon, corresponding to its carcinogenic activity in rats but not in mice (Pereira et al., 2004a). The ability of nongenotoxic carcinogens and tumor promoters to induce renal DNA hypomethylation has not been reported.
Methionine is required for the synthesis of S-adenosylmethionine (SAM), the methyl-group donor for DNA methylation. Methionine has been reported to prevent liver tumorigenesis induced by aflatoxin B1 (Newberne et al., 1990), by diethylnitrosamine followed with promotion by phenobarbital (Fullerton et al., 1990), and more recently DCA (Pereira et al., 2004b). We further demonstrated that methionine prevented DCA-induced DNA hypomethylation, while not affecting other biochemical alterations induced by DCA in the liver (Pereira et al., 2004b), which suggested that DCA-induced DNA hypomethylation was a critical epigenetic alteration required for DCA-induced liver tumors.
In this study, we evaluated chloroform, BDCM, DCA, TCA, and DBA for their ability to induce hypomethylation of DNA and of the c-myc protooncogene in male mouse and rat kidneys as well as the ability of methionine to prevent the hypomethylation in mouse kidney. To further demonstrate the association between DNA hypomethylation and kidney tumors, we determined the ability of chloroform, DCA, and TCA to induce DNA hypomethylation in the kidneys of female B6C3F1 mice that are not susceptible to their renal carcinogenicity.
MATERIALS AND METHODS
Animals and treatment.
Male and female B6C3F1 mice were purchased from Charles River Laboratories (Portage, MI) and maintained as follows, except for the mice and rats used in the BDCM and DBA studies. The animals were housed in the accredited laboratory animal facility at the Medical College of Ohio according to guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care. The mice at 7–8 weeks of age were exposed for a total of 7 days to DCA or TCA with chloroform in their drinking water neutralized with sodium hydroxide to pH 5.0 to 7.0 (Table 1). The concentrations of chloroform, DCA, and TCA were chosen because we had previously demonstrated that the medium to high levels of chloroform prevented DCA, but not TCA, induction of DNA hypomethylation and tumors in mouse liver (Pereira et al., 2001). Some mice with/without administering 3.2 g/l DCA or 4.0 g/l TCA in their drinking water concurrently
The DBA and BDCM studies were performed at Battelle (Columbus, OH) under contracts from the National Institute of Environmental Health Sciences. Male Fischer 344 rats and B6C3F1 mice at 24–30 days of age were obtained from Taconic Laboratory Animals Service (Germantown, NY). At 7–8 weeks of age, the animals in groups of eight were administered BDCM in corn oil by oral gavage at 0, 0.05, and 0.10 g/kg or in their drinking water at 0, 0.35, and 0.70 g/l, or DBA at 0, 1.00, or 2.00 g/l in drinking water neutralized with sodium hydroxide to pH 5.0 to 7.0. The animals were euthanized by carbon dioxide asphyxiation 5 or 7 and 28 days later. At necropsy, the kidneys were collected, rapidly frozen in liquid nitrogen, and stored at –70°C.
Determination of DNA methylation.
DNA was isolated from the kidney by digestion with proteinase K and RNase A followed by organic extraction with phenol, chloroform, and isoamyl alcohol. Methylation of DNA was determined by dot-blot analysis using a monoclonal antibody specifically directed against 5-methylcytosine, as described previously (Tao et al., 2004a,b). Purified DNA (2 μg) was denatured and dotted onto the HybondTM nitrocellulose membranes using a Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was probed with a 1:1,000 dilution of mouse monoclonal antibody specifically against 5-MeC (Eurogentec Company, Belgium), washed with Tris-buffered saline plus Tween 20, pH7.6 (TBST) and subsequently incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary anti-mouse-IgG antibody. The membranes were then treated with enhanced-chemiluminescence Western blotting detection reagents and exposed to Kodak autoradiograph films. Optical density (OD) of the dots was determined using a Scion Image Analysis System (Scion Corp., Frederick, MD). Equal loading of the DNA onto the membrane was indicated by equal intensity of 0.02% methylene blue stained dots.
Methylation of the promoter region of the c-myc gene.
The methylation of the promoter region of the c-myc gene was evaluated using methylation-sensitive restriction endonuclease Hpa II digestion followed by Southern blot analysis, as previously described (Pereira et al., 2001). Isolated DNA was digested overnight with Hpa II. Hpa II does not cut CCGG sites when the internal cytosine is methylated. The digested DNA was electrophoresed and transferred to HybondTM-N+ nylon membranes. The membranes were then hybridized with random 32P-labeled c-myc probe. The c-myc probe was designed from the GeneBank database (GeneBank accession number, M12345) to contain the 1–1315 bp in the promoter region of the gene. The probe was produced by PCR amplification of mouse liver DNA using forward 5'-TCTAGAACCAATGCACAGAGCAAAAG-3' and reverse 5'-GCCTCAGCCCGCAGTCCAGTACTCC-3' primers. The membranes were autoradiographically processed at –70°C with Kodak Biomax MR X-ray film. Optical density of the autoradiograms was measured with the Scion Image Analysis System.
As a control for methylation insensitive digestion, MspI digestion was performed on some of the samples, as previously reported MspI (Pereira et al., 2001; Tao et al., 2002).
Statistical analysis.
The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by the Bonferroni t-test. Statistical significance was indicated by a p-value <0.05.
RESULTS
Effect of Chloroform, DCA, and TCA on Renal DNA Methylation
The ability of chloroform, DCA, and TCA to induce DNA hypomethylation in male mouse kidney is presented in Table 1 and Figure 1. DCA (3.20 g/l) and TCA (4.00 g/l), but not chloroform (1.00 g/l) significantly reduced renal DNA methylation (p < 0.01). DCA and TCA caused 40 and 65% reduction of DNA methylation, respectively. Hence, the order of efficacy to induce renal DNA hypomethylation was TCA > DCA >>> chloroform.
In the kidneys of female mice exposed to chloroform, DCA, and TCA, the optical density of the dots stained for 5-MeC in DNA and its ratio to the density of the dots stained with 0.02% methylene blue for the DNA loading level were not significantly different from the vehicle control mice (p > 0.05). Thus, neither DCA or TCA nor chloroform significantly altered renal DNA methylation in female mouse kidneys (data not shown).
Effect of Chloroform on DCA or TCA-Induced Hypomethylation of the c-myc Gene
The methylation-sensitive restriction endonuclease Hpa II and Southern blot analysis were used to assess the methylation status in the promoter regions of the c-myc gene. 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. A representative autoradiogram and corresponding electrophoresed gel are presented in Figure 2. Treatment with DCA, TCA, and to a lesser extent 1.6 g/liter chloroform result in some of the CCGG sites becoming unmethylated, as evidenced by the appearance of four bands, i.e., 0.2, 0.5, 1.0, and 2.2 kb after digestion with Hpa II. These bands were absent when the DNA was not digested with Hpa II or when it was from control mice. After digestion with methylation-insensitive restriction enzyme MspI, numerous small bands of 100 to 600 bp appeared irrespective of the source of the DNA, indicating that MspI cut most if not all of the 12 CCGG sites in the probed region of the c-myc gene.
It is possible that the lack of bands after Hpa II digestion of DNA from control mice was due to hindered digestion of masked unmethylated CCGG sites. Thus, the presence of bands after Hpa II digestion of DNA from DCA- or TCA-treated mice could result form DNA hypomethylation unmasking unmethylated CCGG sites, allowing them to be digested by Hpa II. To control for this possible situation, we digested the DNA with XbaI and Eco0109I restriction enzyme prior to digestion with Hpa II (Tao et al., 2000b). The region probed for the c-myc gene is franked by an XbaI site at base 2 and an Eco0109I site at base 1490. When DNA from control and treated mice was treated with the two restriction enzymes and probed for the c-myc gene, a band of 1.4 kb was present. When DNA from control mice was digested with XbaI and Eco0109I restriction enzyme either before or after digestion with Hpa II, only the 1.4 kb band was present. Further, when the DNA from treated mice was digested with XbaI and Eco0109I either before or after Hpa II digestion, the 2.2 kb band was no longer present, but instead, a 0.7 kb band appeared along with the other three bands. This would indicate that at least one of the CCGG sites in the 1.4 kb band from treated mice was unmethylated and susceptible to digestion by Hpa II. Hence, the inability of Hpa II to digest the 1.4 kb band from DNA of control mice would indicate that the CCGG sites in this band are methylated.
The effect of coadministering chloroform on the ability of DCA and TCA to decrease the methylation of the c-myc gene is shown in Figures 3 and 4. Chloroform significantly increased the intensity of the four restricted bands in Hpa II-digested renal DNA from DCA-exposed mice in a dose-dependent manner, indicating that chloroform synergically enhanced the ability of DCA to induce the hypomethylation of the c-myc gene (Fig. 3B). In contrast, the intensity of the four bands in Hpa II-digested DNA from TCA-treated mice was not affected by chloroform (Fig. 4B). Thus, chloroform enhanced the ability of DCA, but not TCA, to decrease the methylation of the c-myc gene in male mouse kidney. In comparison, in female mice exposed to DCA or TCA with or without chloroform, the restriction bands were absent after digestion of renal DNA with Hpa II, indicating that neither DCA nor TCA induced hypomethylation of the c-myc gene when coadministered with chloroform.
Effect of Methionine on DCA or TCA-Induced Hypomethylation of the c-myc Gene
The ability of methionine to prevent the hypomethylation of c-myc gene is presented in Figure 5. Bands of 0.5, 1.0, and 2.2 kb were present and used for quantification of the optical density. The 0.2 kb band of Figures 2–4 and 6 was absent in this figure, either because one of the CCGG sites that is cut to result in this band remained methylated or the band was accidentally run off the gel. The densities of the bands were significantly reduced from DCA- or TCA-exposed mice when they were concurrently administered 4.00 or 8.00 g/kg methionine in the diet (Fig. 5B). In fact, when the DCA- or TCA-exposed mice
Effect of DBA on the Methylation of the c-myc Gene and of DNA
Before, evaluating different dose levels of DBA, the time course was determined for it to induce hypomethylation of the c-myc gene in male mouse kidney (Fig. 6). Hpa II-digested DNA from the kidney of male mice exposed to 2.00 g/l of DBA for 7 days contained four restriction bands. These bands were of the same size as the bands from DCA-, TCA-, or chloroform-exposed mice (Fig. 2). The four bands were absent when the DNA was isolated from mice exposed to DBA for 0, 2, or 4 days, demonstrating that between 4 and 7 days of exposure to DBA was required to induce hypomethylation the of c-myc gene in mouse kidney. We have found a similar time course for induction of hypomethylation in mouse liver; that is, 7 but not 4 days of DBA-treatment was sufficient to induce DNA hypomethylation in mouse liver (Tao et al., 2004b).
The ability to induce renal DNA hypomethylation of two dose levels of DBA administered to male mice and rats in their drinking water is presented in Figure 7. Two time points (i.e., 7 and 28 days) were also evaluated, since a longer duration of treatment might be required for lower dose levels of an agent to reach their maximum extent of hypomethylation. Reduction of DNA methylation was significant after 7 days of exposure to 2.00 g/l of DBA and remained suppressed for 28 days. Although 7 days of treatment with the lower concentration of DBA (1.00 g/l) appeared to reduce DNA methylation, it was not significant until 28 days. DBA caused a time- and dose-dependent hypomethylation of renal DNA in mice and rats; methylation was reduced by 40 and 60% in mice and 45 and 56% in rats after 28 days of exposure to 1.00 and 2.00 g/l DBA, respectively.
Effect of BDCM on the Methylation of DNA
BDCM is a drinking water disinfection by-product that induces kidney cancer in male mice and rats, so that it was evaluated for the ability to induce renal DNA hypomethylation in the two species (Figs. 8 and 9). BDCM was administered in the drinking water and by oral gavage to male mice. In mouse kidney, DNA methylation was reduced within 5 to 7 days and without further reduction after 28 days of exposure. The reduction in DNA methylation was dose-dependent and statistically significant, being reduced by 30 and 60% after exposure to the low and high dose of BDCM, respectively (Fig. 8). In rat kidney, DNA methylation was significantly reduced after 5 days of administering 0.10 g/kg BDCM by gavage and remained suppressed for 28 days (Fig. 9). Administering 0.05 g/kg of BDCM did not significantly decrease DNA methylation until 28 days of exposure. After 28 days of exposure, the low and high dose levels of DBCM caused an approximately 30 and 60% reduction in DNA methylation that was similar to the reduction in methylation caused by the low and high dose level of BDCM administered to male mice.
DISCUSSION
Chloroform, BDCM, DBA, DCA, and TCA are commonly found in drinking water as the disinfection by-products of chlorination. Epidemiology studies of chlorination by-products have suggested that cancer of the urinary system is linked to elevated disinfection by-product levels in drinking water (Boorman, 1999; Bull et al., 1995; Koivusalo et al., 1998; Yang et al., 1998). In earlier studies, chloroform and BDCM have been reported to be nephrocarcinogenic in mice and rats (U.S. EPA, 2004a,b). We have demonstrated that TCA promoted male mouse kidney tumors, whereas DCA promoted kidney tumors only when coadministered with chloroform in the drinking water (Pereira et al., 2001). However, few data have been presented that investigate their nephrocarcinogenic mechanism. Therefore, the effect of the chlorination by-products on the methylation of DNA and of the c-myc gene in mouse and rat kidney was determined. Chloroform, BDCM, DBA, DCA, and TCA decreased the methylation of DNA and/or the c-myc gene in mouse and/or rat kidney, indicating that the disinfection by-products have epigenetic activity in the kidney.
Epigenetically, DNA methylation is profoundly altered in carcinogenesis, which include genome-wide hypomethylation and hypermethylation of CpG islands in DNA (Baylin et al., 1998, 2001). DNA hypomethylation is associated with opening of the chromatin configuration and transcriptional activation, leading to chromosomal instability and aberrant expression of genes (Baylin et al., 1998, 2001; Dunn, 2003; Jones and Gonzalgo, 1997). The ability to induce DNA hypomethylation has been proposed as a mechanism for nongenotoxic carcinogens (Counts et al., 1996). We have previously reported that mouse liver tumors, initiated by MNU and promoted by either DCA or TCA, exhibited global hypomethylation in DNA and decreased methylation of protooncogenes including, c-jun, c-myc, and insulin-like growth factor 2 (IGF-II) (Tao et al., 1998, 2000b, 2004a). Furthermore, short-term treatment with either DCA or TCA, as well as many other nongenotoxic carcinogens including trichloroethylene, chloroform, and other trihalomethanes, and peroxisome proliferators have induced hypomethylation of DNA and the c-jun and c-myc genes in mouse liver (Coffin et al., 2000; Ge et al., 2001, 2002; Pereira et al., 2001; Tao et al., 1999, 2000a, 2004b). Additionally, nongenotoxic colon carcinogens, bile acids, rutin, and BDCM have been shown to induce DNA hypomethylation in rat colon (Pereira et al., 2004a). Hence, DNA hypomethylation appears to be a common epigenetic mechanism for nongenotoxic carcinogens.
Although, the ability of nongenotoxic renal carcinogens to induce DNA hypomethylation appears to be related to their carcinogenic activity, the mechanism by which they induce hypomethylation is unknown. Recently, DNA demethylases, including the activity associated with methyl-binding protein-2 (MBD-2) have been identified (Cervoni and Szyf, 2001; Detich, 2003). These enzymes cause the release from 5-MeC of the methyl group as methanol. It is possible that the nongenotoxic carcinogens cause DNA hypomethylation by inducing the DNA demethylase activity. We have preliminary results suggesting that nongenotoxic colon carcinogens (i.e., deoxycholic acid) induced DNA demethylase activity in rat colon. It is also possible that the nongenotoxic carcinogens cause DNA hypomethylation by inducing DNA repair, removal of a piece of DNA containing the 5-MeC, or replication, resulting in unmethylated nascent DNA. However, increased DNA repair would have to be too extensive to account for the 30–50% decrease in DNA methylation; almost all the DNA would have to be repaired. On the other hand, increased DNA replication induced by nongenotoxic carcinogens in mouse liver was consistent with their ability to induce DNA hypomethylation (Ge et al., 2001, 2002).
The drinking water disinfection by-products are nongenotoxic carcinogens with apparently differing potencies in the kidney. Chloroform and BDCM have been reported to be carcinogenic in both mouse and rat kidney, with BDCM apparently being more efficacious (IARC, 1991; Jorgenson et al., 1985; NCI, 1976; NTP, 1987; Roe et al., 1979). Although DCA and TCA do not appear to be carcinogenic in the kidney, TCA has been shown to promote MNU-induced kidney tumors in mice (Pereira et al., 2001), and DBA has yet to be evaluated as a kidney carcinogen. Thus, the relationship between the ability of the five disinfection by-products to induce kidney cancer and their ability to induce DNA hypomethylation was evaluated. In a prior study, we demonstrated that coadministering chloroform prevented DCA- but not TCA-induced promotion of foci and tumors, hypomethylation of DNA, and increase of mRNA expression of the c-myc gene in mouse liver (Pereira et al., 2001). In contrast, in male mice, TCA promoted kidney tumors, while DCA promoted kidney tumors only when coadministered with chloroform in drinking water (Pereira et al., 2001). In the present study, we demonstrated that TCA and DCA caused the hypomethylation of the c-myc gene. Furthermore, coadministering chloroform resulted in enhancement of DCA-induced hypomethylation while not enhancing TCA-induced hypomethylation of the c-myc gene in male mouse kidney. Consequently, the ability of TCA and the synergistic activity of coadministered DCA + chloroform to promote kidney tumors in male mice correlated with their ability to induce DNA hypomethylation.
The relationship between the ability of nongenotoxic carcinogens to induce DNA hypomethylation and cause cancers was also demonstrated by the following results. In female mice, neither DCA nor TCA with/without coadministered chloroform induced hypomethylation, which correlates with their reported inability to promote kidney tumors in female mice (Pereira et al., 2001). In addition, BDCM has been reported to induce colonic DNA hypomethylation in male rats but not in male mice (Pereira et al., 2004), corresponding to its ability to cause colon cancer in male rats but not in male mice (George et al., 2002; U.S. EPA, 2004a; NTP, 1987). Although DBA has not been demonstrated to be carcinogenic, it did induce DNA hypomethylation in the kidney of male mice and rats, suggesting that it might be a kidney carcinogen.
In summary, BDCM, chloroform, DCA, and TCA induced renal DNA hypomethylation, corresponding to their carcinogenic and/or tumor promoting activity in the kidney of mice and rats. The association between the ability to induce DNA hypomethylation and to promote kidney tumors suggests that DNA hypomethylation is involved in the carcinogenic mechanism of these disinfection by-products in the kidney, similar to its apparent involvement in liver and colon carcinogenesis.
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 R03 ES10537 from the National Institute of Environmental Health Sciences, National Institutes of Health. Conflict of interest: none declared.
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Department of Pathology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, Ohio 43614
ABSTRACT
Bromodichloromethane (BDCM), chloroform, dibromoacetic acid (DBA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) are chlorine disinfection by-products (DBPs) found in drinking water that have indicated renal carcinogenic and/or tumor promoting activity. We have reported that the DBPs caused DNA hypomethylation in mouse liver, which correlated with their carcinogenic and tumor promoting activity. In this study, we determined their ability to cause renal DNA hypomethylation. B6C3F1 mice were administered DCA or TCA concurrently with/without chloroform in their drinking water for 7 days. In male, but not female mouse kidney, DCA, TCA, and to a lesser extent, chloroform decreased the methylation of DNA and the c-myc gene. Coadministering chloroform increased DCA but not TCA-induced DNA hypomethylation. DBA and BDCM caused renal DNA hypomethylation in both male B6C3F1 mice and Fischer 344 rats. We have reported that, in mouse liver, methionine prevented DCA- and TCA-induced hypomethylation of the c-myc gene. To determine whether it would also prevent hypomethylation in the kidneys, male mice were administered methionine in their diet concurrently with DCA or TCA in their drinking water. Methionine prevented both DCA- and TCA-induced hypomethylation of the c-myc gene. The ability of the DBPs to cause hypomethylation of DNA and of the c-myc gene correlated with their carcinogenic and tumor promoting activity in mouse and rat kidney, which should be taken into consideration as part of their risk assessment. That methionine prevents DCA- and TCA-induced hypomethylation of the c-myc gene would suggest it could prevent their carcinogenic activity in the kidney.
Key Words: dichloroacetic acid; trichloroacetic acid; DNA hypomethylation; kidney.
INTRODUCTION
Trihalomethanes including chloroform and bromodichloromethane (BDCM) and haloacetic acids including dichloroacetic acid (DCA), trichloroacetic acid (TCA), and dibromoacetic acid (DBA) are major organic disinfection by-products (DBPs) in finished drinking water after chlorination treatment (Chen and Weisel, 1998; Uden and Miller, 1983). The disinfection by-products, with the exception of DBA for which the results of a chronic bioassay has yet to be published, have demonstrated carcinogenic activities in laboratory rodents (U.S. EPA, 2004a–d; IARC, 1991, 2004). Chloroform, BDCM, DCA, and TCA have been demonstrated to be carcinogens and/or tumor promoters in mouse and rat liver (U.S. EPA, 2004a–d; IARC, 1991, 2004; NCI, 1976). The kidney has also been identified as a potential target organ for these disinfection by-products. Chloroform has been reported to induce renal tumors in male mice and rats (Jorgenson et al., 1985; NCI, 1976; Roe et al., 1979). BDCM significantly increased the yield of kidney tumors in male B6C3F1 mice and in both sexes of Fischer 344 rats when administered by gavage (IARC, 1991; NTP, 1987). In N-methyl-N-nitrosourea (MNU)-initiated male, but not female B6C3F1 mice, TCA promoted kidney tumors while DCA did so only when coadministered with chloroform in drinking water (Pereira et al., 2001). DBA has been shown to share numerous biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be carcinogenic in the kidney (Tao et al., 2004b).
Carcinogens are generally considered to increase the risk of cancer by two different mechanisms: genotoxic and epigenetic mechanisms. DNA methylation is a fundamental epigenetic process that not only modulates gene transcription, but is also key to histone acetylation and chromosomal stability. Global hypomethylation of DNA has been proposed to contribute to carcinogenesis (Baylin, 2002; Goodman and Watson, 2002). Hypomethylation of DNA and/or c-myc protooncogene has also been demonstrated in mouse liver and hepatic tumors in response to many nongenotoxic carcinogens, including chloroform, BDCM, DCA, TCA, peroxisome proliferators, and phenobarbital (Coffin et al., 2000; Counts et al., 1996; Ge et al., 2001, 2002; Tao et al., 1998, 2000a,b, 2004a,b). Furthermore, BDCM induces DNA hypomethylation in rat, but not mouse colon, corresponding to its carcinogenic activity in rats but not in mice (Pereira et al., 2004a). The ability of nongenotoxic carcinogens and tumor promoters to induce renal DNA hypomethylation has not been reported.
Methionine is required for the synthesis of S-adenosylmethionine (SAM), the methyl-group donor for DNA methylation. Methionine has been reported to prevent liver tumorigenesis induced by aflatoxin B1 (Newberne et al., 1990), by diethylnitrosamine followed with promotion by phenobarbital (Fullerton et al., 1990), and more recently DCA (Pereira et al., 2004b). We further demonstrated that methionine prevented DCA-induced DNA hypomethylation, while not affecting other biochemical alterations induced by DCA in the liver (Pereira et al., 2004b), which suggested that DCA-induced DNA hypomethylation was a critical epigenetic alteration required for DCA-induced liver tumors.
In this study, we evaluated chloroform, BDCM, DCA, TCA, and DBA for their ability to induce hypomethylation of DNA and of the c-myc protooncogene in male mouse and rat kidneys as well as the ability of methionine to prevent the hypomethylation in mouse kidney. To further demonstrate the association between DNA hypomethylation and kidney tumors, we determined the ability of chloroform, DCA, and TCA to induce DNA hypomethylation in the kidneys of female B6C3F1 mice that are not susceptible to their renal carcinogenicity.
MATERIALS AND METHODS
Animals and treatment.
Male and female B6C3F1 mice were purchased from Charles River Laboratories (Portage, MI) and maintained as follows, except for the mice and rats used in the BDCM and DBA studies. The animals were housed in the accredited laboratory animal facility at the Medical College of Ohio according to guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care. The mice at 7–8 weeks of age were exposed for a total of 7 days to DCA or TCA with chloroform in their drinking water neutralized with sodium hydroxide to pH 5.0 to 7.0 (Table 1). The concentrations of chloroform, DCA, and TCA were chosen because we had previously demonstrated that the medium to high levels of chloroform prevented DCA, but not TCA, induction of DNA hypomethylation and tumors in mouse liver (Pereira et al., 2001). Some mice with/without administering 3.2 g/l DCA or 4.0 g/l TCA in their drinking water concurrently
The DBA and BDCM studies were performed at Battelle (Columbus, OH) under contracts from the National Institute of Environmental Health Sciences. Male Fischer 344 rats and B6C3F1 mice at 24–30 days of age were obtained from Taconic Laboratory Animals Service (Germantown, NY). At 7–8 weeks of age, the animals in groups of eight were administered BDCM in corn oil by oral gavage at 0, 0.05, and 0.10 g/kg or in their drinking water at 0, 0.35, and 0.70 g/l, or DBA at 0, 1.00, or 2.00 g/l in drinking water neutralized with sodium hydroxide to pH 5.0 to 7.0. The animals were euthanized by carbon dioxide asphyxiation 5 or 7 and 28 days later. At necropsy, the kidneys were collected, rapidly frozen in liquid nitrogen, and stored at –70°C.
Determination of DNA methylation.
DNA was isolated from the kidney by digestion with proteinase K and RNase A followed by organic extraction with phenol, chloroform, and isoamyl alcohol. Methylation of DNA was determined by dot-blot analysis using a monoclonal antibody specifically directed against 5-methylcytosine, as described previously (Tao et al., 2004a,b). Purified DNA (2 μg) was denatured and dotted onto the HybondTM nitrocellulose membranes using a Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was probed with a 1:1,000 dilution of mouse monoclonal antibody specifically against 5-MeC (Eurogentec Company, Belgium), washed with Tris-buffered saline plus Tween 20, pH7.6 (TBST) and subsequently incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary anti-mouse-IgG antibody. The membranes were then treated with enhanced-chemiluminescence Western blotting detection reagents and exposed to Kodak autoradiograph films. Optical density (OD) of the dots was determined using a Scion Image Analysis System (Scion Corp., Frederick, MD). Equal loading of the DNA onto the membrane was indicated by equal intensity of 0.02% methylene blue stained dots.
Methylation of the promoter region of the c-myc gene.
The methylation of the promoter region of the c-myc gene was evaluated using methylation-sensitive restriction endonuclease Hpa II digestion followed by Southern blot analysis, as previously described (Pereira et al., 2001). Isolated DNA was digested overnight with Hpa II. Hpa II does not cut CCGG sites when the internal cytosine is methylated. The digested DNA was electrophoresed and transferred to HybondTM-N+ nylon membranes. The membranes were then hybridized with random 32P-labeled c-myc probe. The c-myc probe was designed from the GeneBank database (GeneBank accession number, M12345) to contain the 1–1315 bp in the promoter region of the gene. The probe was produced by PCR amplification of mouse liver DNA using forward 5'-TCTAGAACCAATGCACAGAGCAAAAG-3' and reverse 5'-GCCTCAGCCCGCAGTCCAGTACTCC-3' primers. The membranes were autoradiographically processed at –70°C with Kodak Biomax MR X-ray film. Optical density of the autoradiograms was measured with the Scion Image Analysis System.
As a control for methylation insensitive digestion, MspI digestion was performed on some of the samples, as previously reported MspI (Pereira et al., 2001; Tao et al., 2002).
Statistical analysis.
The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by the Bonferroni t-test. Statistical significance was indicated by a p-value <0.05.
RESULTS
Effect of Chloroform, DCA, and TCA on Renal DNA Methylation
The ability of chloroform, DCA, and TCA to induce DNA hypomethylation in male mouse kidney is presented in Table 1 and Figure 1. DCA (3.20 g/l) and TCA (4.00 g/l), but not chloroform (1.00 g/l) significantly reduced renal DNA methylation (p < 0.01). DCA and TCA caused 40 and 65% reduction of DNA methylation, respectively. Hence, the order of efficacy to induce renal DNA hypomethylation was TCA > DCA >>> chloroform.
In the kidneys of female mice exposed to chloroform, DCA, and TCA, the optical density of the dots stained for 5-MeC in DNA and its ratio to the density of the dots stained with 0.02% methylene blue for the DNA loading level were not significantly different from the vehicle control mice (p > 0.05). Thus, neither DCA or TCA nor chloroform significantly altered renal DNA methylation in female mouse kidneys (data not shown).
Effect of Chloroform on DCA or TCA-Induced Hypomethylation of the c-myc Gene
The methylation-sensitive restriction endonuclease Hpa II and Southern blot analysis were used to assess the methylation status in the promoter regions of the c-myc gene. 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. A representative autoradiogram and corresponding electrophoresed gel are presented in Figure 2. Treatment with DCA, TCA, and to a lesser extent 1.6 g/liter chloroform result in some of the CCGG sites becoming unmethylated, as evidenced by the appearance of four bands, i.e., 0.2, 0.5, 1.0, and 2.2 kb after digestion with Hpa II. These bands were absent when the DNA was not digested with Hpa II or when it was from control mice. After digestion with methylation-insensitive restriction enzyme MspI, numerous small bands of 100 to 600 bp appeared irrespective of the source of the DNA, indicating that MspI cut most if not all of the 12 CCGG sites in the probed region of the c-myc gene.
It is possible that the lack of bands after Hpa II digestion of DNA from control mice was due to hindered digestion of masked unmethylated CCGG sites. Thus, the presence of bands after Hpa II digestion of DNA from DCA- or TCA-treated mice could result form DNA hypomethylation unmasking unmethylated CCGG sites, allowing them to be digested by Hpa II. To control for this possible situation, we digested the DNA with XbaI and Eco0109I restriction enzyme prior to digestion with Hpa II (Tao et al., 2000b). The region probed for the c-myc gene is franked by an XbaI site at base 2 and an Eco0109I site at base 1490. When DNA from control and treated mice was treated with the two restriction enzymes and probed for the c-myc gene, a band of 1.4 kb was present. When DNA from control mice was digested with XbaI and Eco0109I restriction enzyme either before or after digestion with Hpa II, only the 1.4 kb band was present. Further, when the DNA from treated mice was digested with XbaI and Eco0109I either before or after Hpa II digestion, the 2.2 kb band was no longer present, but instead, a 0.7 kb band appeared along with the other three bands. This would indicate that at least one of the CCGG sites in the 1.4 kb band from treated mice was unmethylated and susceptible to digestion by Hpa II. Hence, the inability of Hpa II to digest the 1.4 kb band from DNA of control mice would indicate that the CCGG sites in this band are methylated.
The effect of coadministering chloroform on the ability of DCA and TCA to decrease the methylation of the c-myc gene is shown in Figures 3 and 4. Chloroform significantly increased the intensity of the four restricted bands in Hpa II-digested renal DNA from DCA-exposed mice in a dose-dependent manner, indicating that chloroform synergically enhanced the ability of DCA to induce the hypomethylation of the c-myc gene (Fig. 3B). In contrast, the intensity of the four bands in Hpa II-digested DNA from TCA-treated mice was not affected by chloroform (Fig. 4B). Thus, chloroform enhanced the ability of DCA, but not TCA, to decrease the methylation of the c-myc gene in male mouse kidney. In comparison, in female mice exposed to DCA or TCA with or without chloroform, the restriction bands were absent after digestion of renal DNA with Hpa II, indicating that neither DCA nor TCA induced hypomethylation of the c-myc gene when coadministered with chloroform.
Effect of Methionine on DCA or TCA-Induced Hypomethylation of the c-myc Gene
The ability of methionine to prevent the hypomethylation of c-myc gene is presented in Figure 5. Bands of 0.5, 1.0, and 2.2 kb were present and used for quantification of the optical density. The 0.2 kb band of Figures 2–4 and 6 was absent in this figure, either because one of the CCGG sites that is cut to result in this band remained methylated or the band was accidentally run off the gel. The densities of the bands were significantly reduced from DCA- or TCA-exposed mice when they were concurrently administered 4.00 or 8.00 g/kg methionine in the diet (Fig. 5B). In fact, when the DCA- or TCA-exposed mice
Effect of DBA on the Methylation of the c-myc Gene and of DNA
Before, evaluating different dose levels of DBA, the time course was determined for it to induce hypomethylation of the c-myc gene in male mouse kidney (Fig. 6). Hpa II-digested DNA from the kidney of male mice exposed to 2.00 g/l of DBA for 7 days contained four restriction bands. These bands were of the same size as the bands from DCA-, TCA-, or chloroform-exposed mice (Fig. 2). The four bands were absent when the DNA was isolated from mice exposed to DBA for 0, 2, or 4 days, demonstrating that between 4 and 7 days of exposure to DBA was required to induce hypomethylation the of c-myc gene in mouse kidney. We have found a similar time course for induction of hypomethylation in mouse liver; that is, 7 but not 4 days of DBA-treatment was sufficient to induce DNA hypomethylation in mouse liver (Tao et al., 2004b).
The ability to induce renal DNA hypomethylation of two dose levels of DBA administered to male mice and rats in their drinking water is presented in Figure 7. Two time points (i.e., 7 and 28 days) were also evaluated, since a longer duration of treatment might be required for lower dose levels of an agent to reach their maximum extent of hypomethylation. Reduction of DNA methylation was significant after 7 days of exposure to 2.00 g/l of DBA and remained suppressed for 28 days. Although 7 days of treatment with the lower concentration of DBA (1.00 g/l) appeared to reduce DNA methylation, it was not significant until 28 days. DBA caused a time- and dose-dependent hypomethylation of renal DNA in mice and rats; methylation was reduced by 40 and 60% in mice and 45 and 56% in rats after 28 days of exposure to 1.00 and 2.00 g/l DBA, respectively.
Effect of BDCM on the Methylation of DNA
BDCM is a drinking water disinfection by-product that induces kidney cancer in male mice and rats, so that it was evaluated for the ability to induce renal DNA hypomethylation in the two species (Figs. 8 and 9). BDCM was administered in the drinking water and by oral gavage to male mice. In mouse kidney, DNA methylation was reduced within 5 to 7 days and without further reduction after 28 days of exposure. The reduction in DNA methylation was dose-dependent and statistically significant, being reduced by 30 and 60% after exposure to the low and high dose of BDCM, respectively (Fig. 8). In rat kidney, DNA methylation was significantly reduced after 5 days of administering 0.10 g/kg BDCM by gavage and remained suppressed for 28 days (Fig. 9). Administering 0.05 g/kg of BDCM did not significantly decrease DNA methylation until 28 days of exposure. After 28 days of exposure, the low and high dose levels of DBCM caused an approximately 30 and 60% reduction in DNA methylation that was similar to the reduction in methylation caused by the low and high dose level of BDCM administered to male mice.
DISCUSSION
Chloroform, BDCM, DBA, DCA, and TCA are commonly found in drinking water as the disinfection by-products of chlorination. Epidemiology studies of chlorination by-products have suggested that cancer of the urinary system is linked to elevated disinfection by-product levels in drinking water (Boorman, 1999; Bull et al., 1995; Koivusalo et al., 1998; Yang et al., 1998). In earlier studies, chloroform and BDCM have been reported to be nephrocarcinogenic in mice and rats (U.S. EPA, 2004a,b). We have demonstrated that TCA promoted male mouse kidney tumors, whereas DCA promoted kidney tumors only when coadministered with chloroform in the drinking water (Pereira et al., 2001). However, few data have been presented that investigate their nephrocarcinogenic mechanism. Therefore, the effect of the chlorination by-products on the methylation of DNA and of the c-myc gene in mouse and rat kidney was determined. Chloroform, BDCM, DBA, DCA, and TCA decreased the methylation of DNA and/or the c-myc gene in mouse and/or rat kidney, indicating that the disinfection by-products have epigenetic activity in the kidney.
Epigenetically, DNA methylation is profoundly altered in carcinogenesis, which include genome-wide hypomethylation and hypermethylation of CpG islands in DNA (Baylin et al., 1998, 2001). DNA hypomethylation is associated with opening of the chromatin configuration and transcriptional activation, leading to chromosomal instability and aberrant expression of genes (Baylin et al., 1998, 2001; Dunn, 2003; Jones and Gonzalgo, 1997). The ability to induce DNA hypomethylation has been proposed as a mechanism for nongenotoxic carcinogens (Counts et al., 1996). We have previously reported that mouse liver tumors, initiated by MNU and promoted by either DCA or TCA, exhibited global hypomethylation in DNA and decreased methylation of protooncogenes including, c-jun, c-myc, and insulin-like growth factor 2 (IGF-II) (Tao et al., 1998, 2000b, 2004a). Furthermore, short-term treatment with either DCA or TCA, as well as many other nongenotoxic carcinogens including trichloroethylene, chloroform, and other trihalomethanes, and peroxisome proliferators have induced hypomethylation of DNA and the c-jun and c-myc genes in mouse liver (Coffin et al., 2000; Ge et al., 2001, 2002; Pereira et al., 2001; Tao et al., 1999, 2000a, 2004b). Additionally, nongenotoxic colon carcinogens, bile acids, rutin, and BDCM have been shown to induce DNA hypomethylation in rat colon (Pereira et al., 2004a). Hence, DNA hypomethylation appears to be a common epigenetic mechanism for nongenotoxic carcinogens.
Although, the ability of nongenotoxic renal carcinogens to induce DNA hypomethylation appears to be related to their carcinogenic activity, the mechanism by which they induce hypomethylation is unknown. Recently, DNA demethylases, including the activity associated with methyl-binding protein-2 (MBD-2) have been identified (Cervoni and Szyf, 2001; Detich, 2003). These enzymes cause the release from 5-MeC of the methyl group as methanol. It is possible that the nongenotoxic carcinogens cause DNA hypomethylation by inducing the DNA demethylase activity. We have preliminary results suggesting that nongenotoxic colon carcinogens (i.e., deoxycholic acid) induced DNA demethylase activity in rat colon. It is also possible that the nongenotoxic carcinogens cause DNA hypomethylation by inducing DNA repair, removal of a piece of DNA containing the 5-MeC, or replication, resulting in unmethylated nascent DNA. However, increased DNA repair would have to be too extensive to account for the 30–50% decrease in DNA methylation; almost all the DNA would have to be repaired. On the other hand, increased DNA replication induced by nongenotoxic carcinogens in mouse liver was consistent with their ability to induce DNA hypomethylation (Ge et al., 2001, 2002).
The drinking water disinfection by-products are nongenotoxic carcinogens with apparently differing potencies in the kidney. Chloroform and BDCM have been reported to be carcinogenic in both mouse and rat kidney, with BDCM apparently being more efficacious (IARC, 1991; Jorgenson et al., 1985; NCI, 1976; NTP, 1987; Roe et al., 1979). Although DCA and TCA do not appear to be carcinogenic in the kidney, TCA has been shown to promote MNU-induced kidney tumors in mice (Pereira et al., 2001), and DBA has yet to be evaluated as a kidney carcinogen. Thus, the relationship between the ability of the five disinfection by-products to induce kidney cancer and their ability to induce DNA hypomethylation was evaluated. In a prior study, we demonstrated that coadministering chloroform prevented DCA- but not TCA-induced promotion of foci and tumors, hypomethylation of DNA, and increase of mRNA expression of the c-myc gene in mouse liver (Pereira et al., 2001). In contrast, in male mice, TCA promoted kidney tumors, while DCA promoted kidney tumors only when coadministered with chloroform in drinking water (Pereira et al., 2001). In the present study, we demonstrated that TCA and DCA caused the hypomethylation of the c-myc gene. Furthermore, coadministering chloroform resulted in enhancement of DCA-induced hypomethylation while not enhancing TCA-induced hypomethylation of the c-myc gene in male mouse kidney. Consequently, the ability of TCA and the synergistic activity of coadministered DCA + chloroform to promote kidney tumors in male mice correlated with their ability to induce DNA hypomethylation.
The relationship between the ability of nongenotoxic carcinogens to induce DNA hypomethylation and cause cancers was also demonstrated by the following results. In female mice, neither DCA nor TCA with/without coadministered chloroform induced hypomethylation, which correlates with their reported inability to promote kidney tumors in female mice (Pereira et al., 2001). In addition, BDCM has been reported to induce colonic DNA hypomethylation in male rats but not in male mice (Pereira et al., 2004), corresponding to its ability to cause colon cancer in male rats but not in male mice (George et al., 2002; U.S. EPA, 2004a; NTP, 1987). Although DBA has not been demonstrated to be carcinogenic, it did induce DNA hypomethylation in the kidney of male mice and rats, suggesting that it might be a kidney carcinogen.
In summary, BDCM, chloroform, DCA, and TCA induced renal DNA hypomethylation, corresponding to their carcinogenic and/or tumor promoting activity in the kidney of mice and rats. The association between the ability to induce DNA hypomethylation and to promote kidney tumors suggests that DNA hypomethylation is involved in the carcinogenic mechanism of these disinfection by-products in the kidney, similar to its apparent involvement in liver and colon carcinogenesis.
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 R03 ES10537 from the National Institute of Environmental Health Sciences, National Institutes of Health. Conflict of interest: none declared.
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