2000 Progress 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 Period Covered by this Report: April 20, 2000 through April 19, 2001
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
Objective:The overall objective of the research project is to test the hypothesis that non-genotoxic carcinogens induce cancer by increasing cell proliferation and then decreasing the methylation of the newly synthesized DNA, i.e., 5-methylcytosine (5-MeC). Increased cell proliferation is required to form the hemimethylated DNA requiring methylation. Furthermore, it is hypothesized that the 5-MeC content of DNA is decreased by either reducing the availability of S-adenosyl-l-methionine (SAM) for DNA methylation or by blocking DNA methyltransferase. Specific Aim 1 will evaluate mouse liver and lung non-genotoxic carcinogens including arsenic, chloroprene, dichloroacetic acid, methylene chloride, and tetrachloroethylene for the ability to decrease the methylation of protooncogenes. Specific Aim 2 will determine the mechanism for the decreased methylation of protooncogenes. Specific Aim 3 will demonstrate that an agent that prevents or reverses the hypomethylation of DNA prevents the induction of tumors, supporting the importance of DNA hypomethylation to carcinogenic activity.
Progress Summary:Specific Aim 1 will evaluate mouse liver and lung non-genotoxic carcinogens including arsenic, chloroprene, dichloroacetic acid (DCA), methylene chloride, and tetrachloroethylene for the ability to decrease the methylation of protooncogenes.
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 non-genotoxic carcinogens to cause hypomethylation of another protooncogene, insulin-like growth factor-2 (IGF-2), is being 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 non-carcinogens carcinogens to cause the lost 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, a more universally applicable procedure was developed that allows us to determine the methylation of all genes, as long as their sequence is known, so that the gene can be amplified by PCR. A bisulphite-DNA sequencing procedure has been developed that 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, it was demonstrated that 11 cytosines in the promoter region 2 of the IGF-2 gene are methylated in normal mouse liver. Furthermore, most of these sites were demethylated in liver tumors of B6C3F1 mice promoted by DCA or trichloroacetic acid (TCA).
DNA methylation is one of the mechanisms that control transcription. Thus, we have been determining the relationship between hypomethylation of genes and the expression of their mRNA. Originally, we have been determining the expression of the mRNA of each gene individually by reverse transcriptase-PCR (RT-PCR); however, it was decided the use of a microarray in which the mRNA expression of hundred of genes are simultaneously evaluated would be more cost effective and provide more information. Therefore, we are evaluating different microarrays for determining the effect of the non-genotoxic carcinogens on the expression of different genes. Once we decide on the most appropriate microarray, we will determine the effect of the non-genotoxic carcinogens on mRNA expression of different genes including, but not limited to, those associated with carcinogenesis, cell proliferation, apoptosis, and oxidative damage. This will indicate other genes for which it might be of interest to determine the effect of the non-genotoxic carcinogens on their methylation.
Specific Aim 2 will determine the mechanism for the decreased methylation of protooncogenes. We previously have 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. Prevention of the methylation of hemimethylated sites could result from: (1) decreased activity of DNA Mtase, (2) decreased availability of the methyl donor, SAM, (3) increased concentration of SAH, a product of methylation and an inhibitor of DNA MTase, and (4) blockage of the access of DNA MTase to the hemimethylated sites in DNA. The first three possibilities are unlikely because the hypomethylation induced by DCA, TCA, and Wy-14,643 was not associated with an alteration in liver concentration of either SAM or SAH or with decreased DNA MTase activity. Furthermore, the occurrence of only three bands after Hpa II digestion of DNA from mice treated with DCA and TCA would indicate that only a very few of the 12 CCGG sites in the probed region of the c-myc gene were hypomethylated. Thus, digestion of the DNA with Msp I that also recognizes CCGG site but is not sensitive to methylation at the internal cytosine resulted in numerous smaller bands of 100-600 bp. This indicates that many of the 12 CCGG sites in the promoter region of the c-myc gene remained methylated in the liver of DCA and TCA-treated mice. The specificity of which CCGG sites become hypomethylated could result from DCA and TCA either directly or through a receptor interacting with the chromatin around CCGG sites to block methylation by DNA MTase. We presently are investigating the mechanism by which the non-genotoxic carcinogens might block the accessibility of DNA MTase to CpG sites.
Specific Aim 3 will demonstrate that an agent that prevents or reverses the hypomethylation of DNA prevents the induction of tumors supporting the importance of DNA hypomethylation to carcinogenic activity. We have demonstrated that methionine prevented the hypomethylation of the c-myc gene induced by DCA, TCA, trichloroethylene, and Wy-14,643. Thus, demonstration that the prevention of the hypomethylation is associated with prevention of tumor promotion would support an involvement of DNA hypomethylation in the carcinogenic activity of these chemicals. Prior to performing this cancer prevention study, we are determining the ability of methionine administered in the diet to prevent the hypomethylation induced by DCA without causing toxicity. This study is required because it would be more cost effective and less stressful to the mice if we could administer the methionine in the diet rather than by injection. Once it is demonstrated the methionine can prevent DCA induced hypomethylation of the c-myc and IGF-2 genes, we will proceed with the study to determine whether it is associated with a prevention of DCA promotion of tumors.
Relevance. The expected results of this project should better the understanding of the mechanism of non-genotoxic carcinogens, specifically the involvement of increased cell proliferation and decreased DNA methylation. Using the ability to induce DNA hypomethylation as a biomarker, the dose response relationship of the carcinogens will be determined and extended to dose levels below those applicable in carcinogenesis bioassays. This should decrease the uncertainty in the extrapolation of results from animal carcinogenesis bioassays using high dose levels to the carcinogenic efficacy of more environmentally relevant dose levels.
Future Activities:Future activities include the determination of the ability of arsenic and dimethylarsinic acid to alter the methylation of the c-myc and IGF-2 genes. Unlike the other carcinogens we have so far evaluated, arsenic and its metabolite might cause hypomethylation by decreasing the availability of SAM or inhibiting DNA methyltransferase (DNA MTase). Thus, we will determine the affect of the arsenic compounds on SAM and S-adenosyl-1-homocysteine levels and on DNA MTase activity.
The mechanism by which the carcinogens block the accessibility of DNA MTase to CpG sites and thus prevent methylation is being investigated. First, we are attempting to demonstrate that the mechanism of the carcinogens is, in fact, to block the accessibility of DNA MTase to CpG sites. After this proposed mechanism is confirmed, we will determine how they block the accessibility.
We plan to demonstrate the requirement of DNA hypomethylation for the carcinogenic activity of these agents. This will be accomplished by demonstrating that the prevention of DCA-induced hypomethylation by methionine results in the prevention of tumor promotion by DCA. Specificity of the effect of methionine on DNA methylation will be investigated by determining whether it also prevents DCA enhancement of cell proliferation, glycogen accumulation, oxidative damage, and peroxisome proliferation.
Journal Articles on this Report : 1 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.||