Final Report: Mechanism of Non-genotoxic Occupational CarcinogensEPA Grant Number: R828083
Title: Mechanism of Non-genotoxic Occupational Carcinogens
Investigators: Pereira, Michael A.
Institution: Medical College of Ohio
EPA Project Officer: Hahn, Intaek
Project Period: April 20, 2000 through April 19, 2003
Project Amount: $834,714
RFA: Mechanistic-Based Cancer Risk Assessment Methods (1999) RFA Text | Recipients Lists
Research Category: Health Effects , Human Health , Human Health Risk Assessment , Health
The overall objective of this research project was to test the hypothesis that nongenotoxic environmental carcinogens induce cancer by decreasing the methylation of the newly synthesized DNA (i.e., 5-methylcytosine [5-MeC]). Cell proliferation results in the formation of hemimethylated DNA that then requires methylation. Prevention of the methylation of the hemimethylated DNA results in DNA hypomethylation. DNA hypomethylation is associated with chromosomal instability and rearrangements and with the altered expression of some genes. The specific objectives of this research project were to: (1) evaluate mouse liver and lung nongenotoxic carcinogens including arsenic, dichloroacetic acid (DCA), methylene chloride, tetrachloroethylene (TCE), and trichloroacetic acid (TCA) for the ability to decrease the methylation of DNA; (2) investigate the mechanism by which nongenotoxic carcinogens induce DNA hypomethylation; and (3) demonstrate that DNA hypomethylation induced by nongenotoxic carcinogens is critical for them to induce tumors. This was accomplished by demonstrating that the prevention of carcinogen-induced DNA hypomethylation results in the prevention of tumors induced by the carcinogen. We determined the ability of methionine to prevent DNA hypomethylation induced by two environmental carcinogens?the drinking water disinfection byproduct DCA, and the drinking water contaminant arsenic. The prevention of DNA hypomethylation results in the prevention of tumors induced by these two carcinogens. This demonstrates that DNA hypomethylation is critical to the carcinogenic activity of these two important environmental carcinogens.
Objective 1: Evaluation of Mouse Liver and Lung Nongenotoxic Carcinogens Including Arsenic, DCA, Dibromoacetic Acid, Methylene Chloride, TCE, and TCA for the Ability to Decrease the Methylation of DNA
We have demonstrated that methylene chloride administered by inhalation decreased the methylation of the c-myc gene in the lung, liver, kidney, and bladder of mice. Thus, it was demonstrated that administering the carcinogens by inhalation causes hypomethylation in not only the lung, but in other systemic organs that are targets for carcinogenic activity.
The ability of the nongenotoxic carcinogens to cause hypomethylation of another protooncogene, insulinlike growth factor-2 (IGF-2), was determined. IGF-2 is being investigated because it is imprinted by methylation of its promoter region, so that it allows the determination of the ability of the noncarcinogens to cause the loss of imprinting. Also, the Hpa II digestion of the DNA procedure used for c-myc cannot be used for IGF-2 due to the lack of CCGG sites. Therefore, another procedure was used that allowed us to determine the methylation of a gene as long as its sequence is known so that the gene can be amplified by polymerase chain reaction (PCR). The method being used is the bisulphite-DNA sequencing procedure. The procedure consists of bisulphite treatment, PCR amplification, cloning of the PCR product, and sequencing of the clones to determine the methylation status in the promoter 2 of mouse IGF-2 gene. Using this procedure, the methylation status of 28 CpG sites in the differentially methylated region-2 (DMR-2) of mouse IGF-2 gene was determined. In mouse liver, 79.3 ± 1.7 percent of the sites were methylated, while in tumors from DCA and TCA-treated mice, only 8.7 ± 2.6 percent and 10.7 ± 7.4 percent of the sites were methylated, respectively. A few of the tumors had methylation at CpNpG sites that were not found in noninvolved liver. The mRNA expression of the IGF-2 gene was increased in DCA and TCA-promoted liver tumors relative to noninvolved liver. The results support the involvement of the hypomethylation of DNA and of the IGF-2 gene in DCA- and TCA-promoted hepatocarcinogenesis.
Both dimethylarsinic acid and sodium arsenite decreased the methylation of the IGF-2 gene. However, because IGF-2 is imprinted, the two chromatids of the gene have different patterns of methylation. This hampered the interpretation of our results. This was not a problem, however, when we investigated tumors induced by arsenic because in these tumors, both chromatids have a very low level of methylation.
Due to the problem of using decreased methylation of the IGF-2 gene as an indicator of DNA methylation in liver tissue, we have developed another procedure to determine the extent of DNA methylation in liver. This procedure is dot blot analysis using an antibody specific to 5-MeC. This procedure has the advantage that it is not dependent on a specific gene. It also can be used to detect the level of DNA methylation in tissue sections. Basically, the procedure involves isolating DNA, immobilizing it onto a membrane, and then determining the level of 5-MeC by immunohistochemistry using the antibody to 5-MeC. The antibody was specific to 5-MeC and did not interact with unmethylated cytosine and the reaction with DNA was proportional to the level of 5-MeC in DNA and was sensitive enough to determine the level of methylation of 0.5 mg DNA. Using the dot blot analysis with the 5-MeC antibody, the DNA in DCA and TCA-promoted tumors was hypomethylated. We also have used this procedure to determine the level of DNA methylation in the studies discussed under the third objective, which determined the ability of methionine to prevent dichloroacetic acid, arsenic-induced liver and lung tumors in mice.
DNA methylation is one of the mechanisms that controls transcription. DNA methylation also is involved in chromosomal stability. Thus, DNA hypomethylation induced by nongenotoxic carcinogens could result in chromosome instability, exchanges, deletions, and loss. This will increase the probability of cancer. DNA hypomethylation also has been associated with the hypermethylation of promoter regions of tumor suppressor genes. This decreases the expression of these cancer-related genes; this is another way that DNA hypomethylation induced by nongenotoxic carcinogens can cause cancer.
Objective 2: Investigation of the Mechanism by which Nongenotoxic Carcinogens Induce DNA Hypomethylation
We have previously reported that chloroform, DCA, TCA, and Wy-14,643 (a peroxisome proliferator) decreased the methylation of DNA and of the promoter region of the c-myc gene. These agents appear to cause hypomethylation of the c-myc gene by preventing the methylation of hemimethylated DNA formed when DNA is replicated. However, the liver has a very low level of DNA replication, so that DCA and TCA would have to increase cell proliferation and DNA replication for there to be hemimethylated sites requiring methylation. Thus, we have demonstrated that DCA, TCA, and Wy-14,643 increased cell proliferation and DNA replication before they induced hypomethylation of the c-myc gene.
The ability of dichloroacetic acid to induce DNA hypomethylation was not associated with many of its other activities. Thus, methionine prevented DCA from inducing the hypomethylation of DNA and of the c-myc and IGF-II genes without altering its ability to increase the weight of the liver, to induce cell proliferation, to increase the level of glycogen, to cause oxidative damage or to cause peroxisome proliferation. This indicated that DNA hypomethylation is induced by DCA independent of many of its other activities.
Objective 3: Demonstration that DNA Hypomethylation Induced by Nongenotoxic Carcinogens Is Critical for Them to Induce Tumors
We started a study to determine whether methionine prevention of DNA hypomethylation induced by DCA resulted in the prevention of DCA-promotion of N-methyl-N-nitrosourea (MNU)-initiated liver tumors in mice. However, the MNU induced numerous lymphomas that occurred early in the study. Although we had found MNU-induced lymphomas in previous studies, their incidence was less than 5 percent and occurred at around 1 year. In this study, the lymphomas were present by 2 months, so the study was terminated. therefore, we performed a study in which we did not initiate the mice with MNU, but rather determined the effect of methionine on DCA-induced liver tumors in mice that were not initiated with a carcinogen.
Female B6C3F1 mice were administered 3.2 g/L DCA in their drinking water with 0, 4, or 8 g/kg methionine added to their diet. Mice were sacrificed at 4, 8, and 44 weeks after administering DCA. DCA increased the liver/body weight ratio and in the liver, increased the level of glycogen, peroxisomes, and oxidative damage at 4 and/or 8 weeks. At the two early sacrifices, DCA decreased the methylation of DNA and the c-myc gene. Of all these activities of DCA at 4 and 8 weeks, the hypomethylation of DNA and the c-myc gene was prevented by methionine. At 44 weeks, 4 and 8 g/kg methionine decreased the multiplicity of liver tumors by 87 and 98 percent, respectively. Thus, in the absence of methionine, the multiplicity of liver tumors was 1.28 tumors/mouse, while in the presence of 4 or 8 g/kg methionine, it was 0.167 and 0.0278. In contrast, the yield of altered hepatocyte foci was increased by 4 g/kg methionine from 2.41 to 3.4 foci/mouse. Methionine at 8 g/kg decreased the yield of foci to 0.939 foci/mouse. This would suggest that methionine decreased the yield of liver tumors by slowing the progression of foci to tumors. These results also demonstrated that the ability of DCA to cause DNA hypomethylation is critical for it to cause liver tumors, because hypomethylation was the only DCA-induced alteration that was prevented by methionine when it prevented liver tumors.
The ability of methionine to prevent dimethylarsinic acid and sodium arsenite to induce lung tumors in Strain A mice was determined. Although mice administered both forms of arsenic (100 and 200 mg/kg sodium arsenite and 400 and 800 mg/kg dimethylarsinic acid) had increased yield of lung tumors, the increase was not statistically significant. Thus, we failed to demonstrate that either form of arsenic induced lung tumors in mice. Methionine added to the diet at 8 g/kg decreased the yield of lung tumors in mice not administered arsenic as well as in mice administered dimethylarsinic acid or sodium arsenite. This would suggest that DNA hypomethylation is important for the development of spontaneous lung tumors in mice.
The effect of chloroform on DCA and TCA-induced hypomethylation and expression of the c-myc gene and on their promotion of liver and kidney tumors was determined. B6C3F1 mice were administered 0, 400, 800, and 1,600 mg/L chloroform in drinking water and 500 mg/kg DCA or TCA administered daily by gavage. DCA, TCA, and to a lesser extent, chloroform, decreased the methylation and increased the mRNA expression of the c-myc gene. Co-administering chloroform prevented only DCA and not TCA-induced hypomethylation and increased mRNA expression of the gene. The effect of chloroform on tumor promotion by DCA and TCA was determined in female and male B6C3F1 mice initiated on day 15 of age with MNU. Starting at 5 weeks of age, the mice received, in their drinking water, DCA (3.2 g/L) or TCA (4.0 g/L) with 0, 800, or 1,600 mg/L chloroform until sacrificed at 36 weeks. Liver tumors promoted by DCA and TCA were predominantly basophilic except for DCA-treated female mice that were eosinophilic. Only DCA promoted foci of altered hepatocytes and they were eosinophilic in both sexes. Chloroform prevented DCA, but not TCA promotion of liver foci and tumors. In male mice, TCA promoted kidney tumors, while DCA promoted kidney tumors only when co-administered with chloroform. Hence, chloroform prevented the hypomethylation and increased mRNA expression of the c-myc gene and the promotion of liver tumors by DCA, while enhancing DCA-promotion of kidney tumors. Thus, the concurrent exposure to two carcinogens, chloroform and DCA, resulted in less than additive activity in one organ and synergism in another organ.
The results of this project have improved the understanding of the mechanism of nongenotoxic carcinogens. The project resulted in the demonstration that the ability of dichloroacetic acid to produce liver tumors in mice required that it caused DNA hypomethylation. Hence, we demonstrated that methionine prevented DCA from causing both DNA hypomethylation and liver tumors in mice. Methionine also prevented spontaneous lung tumors in mice. The interaction of two carcinogens, chloroform and DCA, was complex and related to their interaction with respect to DNA hypomethylation. Chloroform prevented both the hypomethylation of the c-myc gene and the promotion of liver tumors by DCA. In contrast, in the mouse kidney, chloroform enhanced DCA-induction of DNA hypomethylation and its promotion of kidney tumors. In summary, we demonstrated that DNA hypomethylation was critical both for DCA to cause liver tumors and to promote kidney tumors in mice and for the occurrence of spontaneous lung tumors in mice. Many important environmental nongenotoxic carcinogens including DCA, chloroform, other trihalomethanes, TCA, and numerous peroxisome proliferators have been shown to cause DNA hypomethylation. Furthermore, we have demonstrated under this grant that three other environmental carcinogens, dibromoacetic acid, methylene chloride, and tetrachloroethylene also caused DNA hypomethylation. This would suggest that the ability to induce DNA hypomethylation also might be critical for the carcinogenic activity of these numerous important environmental carcinogens. Our results also indicate that the ability to induce DNA hypomethylation could be developed as a biomarker and used to determine dose-response relationship for nongenotoxic carcinogens, interactions between environmental carcinogens, and for interspecies extrapolation. This could decrease the uncertainty in the extrapolation of the results obtained from animal carcinogenesis bioassays to the estimation of the carcinogenic efficacy of more environmentally relevant dose levels.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
|Other project views:||All 5 publications||3 publications in selected types||All 3 journal articles|
||Pereira MA, Kramer PM, Conran PB, Tao L. Effect of chloroform on dichloroacetic acid and trichloroacetic acid-induced hypomethylation and expression of the c-myc gene and on their promotion of liver and kidney tumors in mice. Carcinogenesis 2001;22(9):1511-1519.||
||Tao L, Wang W, Bridges AS, Li L, Yang S, Kramer PM, Pereira MA. Effect of dibromoacetic acid in drinking water on the DNA methylation in mice and rats. Toxicological Sciences 2004;82(1):62-69.||
||Tao L, Li Y, Kramer PM, Wang W, Pereira MA. Hypomethylation of DNA and the insulin-like growth factor-II gene in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors. Toxicology 2004;196(1-2):127-36.||