ToxSci Advance Access originally published online on December 2, 2003
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Toxicological Sciences 77, 243-248 (2004)
Copyright © 2004 by the Society of Toxicology
CARCINOGENICITY |
Prevention by Methionine of Dichloroacetic Acid–Induced Liver Cancer and DNA Hypomethylation in Mice
* Department of Pathology, Medical College of Ohio, Toledo, Ohio 43614
Received July 29, 2003; accepted October 22, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dichloroacetic acid (DCA) is a liver carcinogen that induces DNA hypomethylation in mouse liver. To test the involvement of DNA hypomethylation in the carcinogenic activity of DCA, we determined the effect of methionine on both activities. Female B6C3F1 mice were administered 3.2 g/l DCA in their drinking water and 0, 4.0, and 8.0 g/kg methionine in their diet. Mice were sacrificed after 8 and 44 weeks of exposure. After 8 weeks of exposure, DCA increased the liver/body weight ratio and caused DNA hypomethylation, glycogen accumulation, and peroxisome proliferation. Methionine prevented completely the DNA hypomethylation, reduced by only 25% the glycogen accumulation, and did not alter the increased liver/body weight ratio and the proliferation of peroxisomes induced by DCA. After 44 weeks of exposure, DCA induced foci of altered hepatocytes and hepatocellular adenomas. The multiplicity of foci of altered hepatocytes/mouse was increased from 2.41 ± 0.38 to 3.40 ± 0.46 by 4.0 g/kg methionine and decreased to 0.94 ± 0.24 by 8.0 g/kg methionine, suggesting that methionine slowed the progression of foci to tumors. The low and high concentrations of methionine reduced the multiplicity of liver tumors/mouse from 1.28 ± 0.31 to 0.167 ± 0.093 and 0.028 ± 0.028 (i.e., by 87 and 98%, respectively). Thus, the prevention of liver tumors by methionine was associated with its prevention of DNA hypomethylation, indicating that DNA hypomethylation was critical for the carcinogenic activity of DCA.
Key Words: chemoprevention; dichloroacetic acid; DNA hypomethylation; methionine; mouse liver tumors; prevention of cancer.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dichloroacetic acid (DCA) is found in finished drinking water being formed as a by-product of chlorine disinfection (Boorman, 1999
; Chen and Weisel, 1998; Uden and Miller, 1983). DCA has been shown to induce hepatocellular adenomas and carcinomas in mice (Boorman, 1999; Bull et al., 1990; Herren-Freund et al., 1987; Pereira, 1996). It has also been shown to promote N-methyl-N-nitrosourea– (MNU-) initiated foci of altered hepatocytes and liver tumors in B6C3F1 mice (Pereira and Phelps, 1996; Pereira et al., 1997).
We have shown that DCA induced hypomethylation of DNA and the c-jun and c-myc protooncogenes in mouse liver that could be involved in its carcinogenic activity (Ge et al., 2001b; Pereira et al., 2001; Tao et al., 2000a,b). DNA methylation is a naturally occurring modification of DNA that involves an addition of a methyl group to the 5-position carbon of the cytosine ring to form 5-methylcytosine (5-MeC). DNA hypomethylation is a common event in most cancers including liver cancer and has been proposed to be involved in chemical carcinogenesis (Baylin, 2002; Counts and Goodman, 1995; Goodman and Watson, 2002; Razin and Shemer, 1999). Liver tumors induced by a choline-methionine–deficient diet in both mice and rats exhibited DNA hypomethylation and decreased methylation of protooncogenes including H-ras, c-myc, and c-fos (Counts et al., 1996; Henning and Swendseid, 1996; Wainfan and Poirier, 1992). We have demonstrated that the protooncogenes c-jun and c-myc are hypomethylated in mouse liver tumors initiated by MNU and promoted by either DCA or trichloroacetic acid (TCA; Tao et al., 1998, 2000b). Liver tumors induced by phenobarbital have also been reported to contain hypomethylated protooncogenes (Counts et al., 1997). Short-term treatment with chloroacetic acids, phenobarbital, and other nongenotoxic carcinogens, including trichloroethylene, chloroform, other trihalomethanes, and peroxisome proliferators, induced hypomethylation of DNA and protooncogenes in mouse liver (Coffin et al., 2000; Counts et al., 1996, 1997; Ge et al., 2001a, 2002; Tao et al., 1998, 1999, 2000b). Furthermore, we have shown that methionine could prevent hypomethylation induced by some of these carcinogens (Ge et al., 2001a; Tao et al., 2000a). Methionine administered concurrently with DCA, TCA, trichloroethylene, or Wy 14,643 prevented their ability to cause the hypomethylation of DNA and of the protooncogenes c-jun and c-myc. Hence, if DNA hypomethylation is critical for the carcinogenic mechanism of DCA, then its prevention by methionine should result in the prevention of liver tumors, which is what we tested.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and treatment.
Female B6C3F1 mice were purchased from Charles River Laboratories (Portage, MI). The mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care– (AAALAC-) accredited laboratory animal facility of the Medical College of Ohio. They were maintained in polycarbonate shoebox cages with Anderson Bed-o-Cob 1/8 bedding (Andersons, Maumee, OH). The mice received AIN-76A diet consisting of casein (20%), DL-methionine (0.3%), cornstarch (52%), dextrose (13%), corn oil (5%), alphacel (5%), AIN mineral mixture 3 (5%), AIN vitamin mixture (1.0%), and choline bitartrate 0.2% (Dyets, Inc., Bethlehem, PA).
When the mice were 7 to 8 weeks old, they were divided into the exposure groups presented in Table 1. They then started to receive 3.2 g/l DCA (Aldrich Chemical Co., Milwaukee, WI) neutralized to pH 6–7 with sodium hydroxide in their drinking water and 4.0 or 8.0 g/kg of L-methionine (Sigma Chemical Co., St. Louis, MO) added to their diet. The methionine content of the AIN-76A diet was 7.3 g/kg so that the additional methionine increased the concentration to 11.3 and 15.3 g/kg. Methionine was administered in the diet so that the mice would be exposed to it at the same time that they drank the water containing DCA. The diet and drinking water were provided ad libitum. Mice were sacrificed by carbon dioxide asphyxiation after 8 and 44 weeks of exposure to DCA.
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Body and liver weights were obtained at necropsy and the liver was evaluated for tumors. The whole liver was cut into blocks of 3 to 4 mm each. The blocks were fixed in phosphate-buffered formalin and embedded in paraffin. Then, 5-µm sections were stained with hematoxylin and eosin and evaluated for foci of altered hepatocytes, hepatocellular adenomas, and hepatocellular adenocarcinomas. Foci of altered hepatocytes contained six or more cells and hepatocellular adenomas were distinguished from foci by the occurrence of compression at greater than 75% of their border.
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 at 490 nm (Lo et al., 1970) and was expressed as µg glycogen/mg 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 (Poosch and Yamazaki, 1986). Liver was homogenated in 60 mM potassium phosphate (pH 7.0) containing 0.2% Triton X-100 and centrifuged at 1000 x g. The 1000 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 horseradish peroxidase, 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.
DNA-dot blot analysis of 5-MeC in DNA.
DNA was isolated from liver by digestion with RNase A and proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol (Tao et al., 2000a). Purified DNA (2 µg) was denatured with 0.1 N NaOH at 100°C for 5 min, neutralized with 2 M ammonium acetate, and then dotted onto a HybondTM nitrocellulose membrane using a Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was baked under vacuum at 80°C for 2 h to fix the DNA to the membrane. It was then incubated in 5% fat-free powdered milk dissolved in Tris-buffered saline + Tween-20 (TBST) blocking solution (pH 7.6) for 2 h. The membrane was incubated with a 1:1000 dilution of mouse monoclonal primary antibody specific against 5-MeC (Eurogentec, Seraing, Belgium) for 2 h, washed with TBST (pH 7.6), and subsequently incubated with a 1:2000 dilution of horseradish peroxidase–conjugated secondary antimouse-IgG antibody for 1 h. The membranes were washed again with TBST (pH 7.6), treated with enhanced chemiluminescence Western blotting detection reagents, and exposed to Kodak autoradiograph films (Eastman Kodak Co., Rochester, NY). Optical density of the dots was measured with the Scion Image Analysis System (Scion Corp., Frederick, MD). Equal loading of the DNA onto the membrane was indicated by equal intensity of methylene blue stained dots. The optimum dilution of the 5-MeC antibody was previously determined to be 1:1000 using 1:500–1:2000 dilutions. Specificity of the anti–5-MeC antibody was demonstrated by the ability of 5-MeC but not cytosine to block the antibody.
Statistical analysis.
Results were analyzed by a one-way ANOVA followed by the Bonferroni t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Effect of Methionine on DCA-Induced Liver Foci and Tumors
Mortality during the study was limited to three mice that received DCA + 8.0 g/kg methionine and one mouse each that received DCA + 4.0 g/kg methionine and 8.0 g/kg methionine without DCA. There was an increase in body weight during weeks 12 to 36 in the mice that received 8.0 g/kg methionine without DCA. There was no other treatment-related alteration in body weight. Also, there was no treatment-related effect on diet consumption.
Foci of altered hepatocytes and hepatocellular adenomas were found at the week 44 sacrifice. The effect of methionine on DCA-induced foci of altered hepatocytes and hepatocellular adenomas is presented in Figure 1. DCA induced a significant number of foci and adenomas, that is 2.41 ± 0.38 foci/mouse and 1.28 ± 0.31 adenomas/mouse compared to zero foci and adenomas in 16 mice that were not administered DCA. The foci and adenomas stained eosinophilic with relatively large hepatocytes and nuclei, as reported previously for DCA-induced neoplastic lesions in female mice (Latendresse and Pereira, 1997; Pereira, 1996; Pereira et al., 2001). The addition of 4.0 or 8.0 g/kg methionine to the AIN-76A diet significantly reduced the multiplicity of adenomas/mouse to 0.167 ± 0.093 and 0.028 ± 0.028, respectively. In contrast, the addition of 4.0 g/kg methionine did not reduce the multiplicity of foci/mouse but rather increased it to 3.4 ± 0.46 (not statistically significant). The addition of 8.0 g/kg methionine did reduce the yield to 0.94 ± 0.24 foci/mouse. There were no foci or tumors in 16 mice that received either the control diet or 8.0 g/kg methionine without DCA.
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The Effect of Methionine on DCA Enhancement of Liver Weight and Glycogen
DCA increased the liver/body weight ratio at 8 and 44 weeks (Fig. 2
). Neither 4.0 nor 8.0 g/kg methionine prevented a DCA-induced increase in liver weight. Methionine (8.0 g/kg) did not alter the liver weight of mice that did not receive DCA. A DCA-induced increase in liver weight has been associated with an increase in liver glycogen (Bull et al., 1990). After 8 weeks of exposure to DCA, liver glycogen increased 120% from 52.4 ± 2.38 to 115.7 ± 3.46 mg/g liver (Fig. 3). Both concentrations of methionine decreased liver glycogen in mice exposed to DCA by approximately 25% to 91.3 ± 2.90 and 86.0 ± 2.01 mg/g liver for 4.0 and 8.0 mg/kg methionine, respectively. However, liver glycogen was still significantly increased by approximately 55% relative both to control mice and to mice that received 8.0 g/kg methionine without DCA. Methionine did not alter liver glycogen in mice that did not receive DCA.
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The Effect of Methionine on Peroxisome Proliferation Induced by DCA
Exposure to DCA resulted in peroxisome proliferation as measured by lauroyl-CoA oxidase activity (Fig. 4
). Neither concentration of methionine affected the level of peroxisome proliferation induced by DCA. Furthermore, 8.0 g/kg methionine did not alter lauroyl-CoA oxidase activity in mice that did not receive DCA.
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The Effect of Methionine on DNA Hypomethylation Induced by DCA
DCA reduced the level of DNA methylation in the liver; in mice that did not receive DCA, methionine (8.0 g/kg) increased the level of methylation (Fig. 5
). Both concentrations of methionine prevented DCA-induced DNA hypomethylation. In fact, 8.0 g/kg methionine increased DNA methylation in mice administered DCA to a level significantly greater than that in control mice. Furthermore, DNA methylation was increased to the same level by 8.0 g/kg methionine in control mice and in mice administered DCA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCA is found in drinking water as a by-product of chlorine disinfection (Boorman, 1999
; Chen and Weisel, 1998; Uden and Miller, 1983) and is a metabolite of the important solvent and degreaser trichloroethylene (International Agency for Research on Cancer, 1995). DCA is a mouse liver carcinogen and tumor promoter for which a nongenotoxic or epigenetic mechanism has been proposed (Boorman, 1999; Bull et al., 1990; Herren-Freund et al., 1987; Pereira, 1996; Pereira and Phelps, 1996; Pereira et al., 1997). One epigenetic mechanism proposed for nongenotoxic carcinogens is the induction of DNA hypomethylation. DNA hypomethylation has been associated with numerous molecular alterations that can enhance the progression of cancer including chromosomal and genetic instability, decreased histone deacetylase activity, and increased expression of genes (Baylin, 2002; Kress et al., 2001; Li et al., 2003; Recillas-Targa, 2002; Robertson and Jones, 2000). We have previously reported that DCA decreased the methylation of DNA and the c-jun and c-myc protooncogenes (Ge et al., 2001b; Pereira et al., 2001; Tao et al., 2000a,b).
The relationship between DCA-induced DNA hypomethylated and its carcinogenic activity was evaluated by determining the ability of methionine to prevent both hypomethylation and tumors. One mechanism by which carcinogens could cause DNA hypomethylation is decreasing the availability of s-adenosyl methionine (SAM), the methyl donor for methylation. Since methionine increases the liver content of SAM, we previously investigated its ability to prevent carcinogen-induced DNA hypomethylation. These studies demonstrated that methionine did prevent the hypomethylation of DNA and protooncogenes induced by DCA, TCA, trichloroethylene, and another peroxisome proliferator, Wy-14,643, in the liver (Ge et al., 2001a; Tao et al., 2000a). Here we demonstrated that the prevention of DCA-induced DNA hypomethylation was associated with the prevention of liver tumors. The ability of methionine to prevent DCA-induced DNA hypomethylation and liver tumors was similar, both being almost completely prevented by the high concentration of methionine. This is the first study to demonstrate that the prevention of carcinogen-induced DNA hypomethylation correlated with the prevention of liver tumors. AIN-76A diet contains approximately 7.3 g/kg including 3 g/kg of added DL-methionine. In other unpublished studies, we have administered AIN-76A diet without the added 3 g/kg methionine to mice without any effect on body weight or clinical signs of toxicity. Therefore, lowering the methionine content of the AIN-76A diet would be expected to increase the yield of DCA-induced liver tumors.
In contrast to methionine prevention of DNA hypomethylation and liver tumors, it either did not prevent or prevented to a lesser extent other effects of DCA in the liver. Methionine did not prevent DCA-induced hepatomegaly and peroxisome proliferation. DCA-induced glycogen accumulation was prevented by methionine to a much lesser extent than liver tumors; that is, glycogen accumulation was only reduced by 25%, while DNA hypomethylation and liver tumors were almost completely prevented. Also, 8 weeks of exposure to DCA did not result in a significant increase in cell proliferation, as measured by the Proliferating Cell Nuclear Antigen-Labeling Index (data not shown). This is consistent with previous reports stating that 2 or more weeks of exposure to DCA did not significantly enhance cell proliferation in mouse liver (Bull, 2000; DeAngelo et al., 1999; Pereira, 1996; Sanchez and Bull, 1990). We have shown that, although methionine prevented DNA hypomethylation induced by Wy-14,643, it increased cell proliferation in the liver (Ge et al., 2001b). Therefore, the prevention of DCA-induced liver tumors by methionine is not likely the result of preventing DCA enhancement of cell proliferation. The correlation between the prevention by methionine of DCA-induced liver tumors and DNA hypomethylation, but not the other effects of DCA, suggests that DNA hypomethylation is critical for the carcinogenic activity of DCA.
Methionine affected differently the ability of DCA to induce foci of altered hepatocytes and liver tumors. Liver tumors were prevented by both 4.0 and 8.0 g/kg methionine added to the diet. However, only the higher concentration of 8.0 g/kg methionine prevented foci of altered hepatocytes. While 4.0 g/kg methionine decreased the multiplicity of liver tumors by approximately 90%, the multiplicity of foci was increased, although not to the point of being statistically significant. This would suggest that the mode of action of methionine is to slow the neoplastic progression of foci to tumors.
Other mouse liver carcinogens including TCA, trihalomethanes including chloroform, phenobarbital, and numerous peroxisome proliferators have been reported to cause DNA hypomethylation (Coffin et al., 2000; Counts and Goodman, 1995; Counts et al., 1996, 1997; Ge et al., 2001a, 2002; Tao et al., 1998, 1999, 2000b). Our previous results also suggested a threshold for DNA hypomethylation induced by DCA, TCA, trichloroethylene, chloroform, and Wy-14,643 (Coffin et al., 2000; Ge et al., 2001a,b). We have also shown that methionine prevented DNA hypomethylation induced by DCA, TCA, trichloroethylene, and Wy 14,643 (Ge et al., 2001a; Tao et al., 2000a). We reported here that methionine prevented DCA-induced liver tumors. Methionine has also been reported to prevent the promotion by phenobarbital of liver carcinomas initiated by diethylnitrosamine in C3H mice (Fullerton et al., 1990). Therefore, it would be of interest to determine whether methionine would prevent DNA hypomethylation induced by phenobarbital as well as DNA hypomethylation and liver tumors induced by other nongenotoxic carcinogens.
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ACKNOWLEDGMENTS |
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This research was supported by grant R82808301 from the U.S. Environmental Protection Agency’s Science to Achieve Results (STAR) program.
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NOTES |
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1 To whom correspondence should be addressed at Ohio State University, College of Medicine and Public Health, Department of Internal Medicine, Division of Hematology and Oncology, 300 West Tenth Avenue, 1148 CHRI, Columbus, OH 43210. Fax: (614) 293-3333. E-mail: pereira.25{at}osu.edu.
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L. Tao, W. Wang, L. Li, P. M. Kramer, and M. A. Pereira Effect of Dibromoacetic Acid on DNA Methylation, Glycogen Accumulation, and Peroxisome Proliferation in Mouse and Rat Liver Toxicol. Sci., November 1, 2004; 82(1): 62 - 69. [Abstract] [Full Text] [PDF]
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