* Department of Internal Medicine, Division of Hematology and Oncology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210, and Department of Pathology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, Ohio 43614
1 To whom correspondence should be addressed at The Ohio State University, Comprehensive Cancer Center, CCC-363, Room 301, 410 W. 12th Avenue, Columbus, OH 43210. Fax: (614) 292-8893. E-mail: tao-2{at}medctr.osu.edu.
Received March 11, 2005; accepted July 7, 2005
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ABSTRACT |
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Key Words: dichloroacetic acid; trichloroacetic acid; DNA hypomethylation; kidney.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 11315 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.
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RESULTS |
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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.
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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.
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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.
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DISCUSSION |
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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 3050% 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.
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ACKNOWLEDGMENTS |
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