2001 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, 2001 through April 19, 2002
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 is to test the hypothesis that non-genotoxic carcinogens induce cancer by decreasing the methylation of the newly synthesized DNA (i.e., 5-methylcytosine (5-MeC)) and then increase the promoter of tumor suppressor genes to decrease their expression. 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. We will evaluate mouse liver and lung non-genotoxic carcinogens including arsenic, dichloroacetic acid, methylene chloride, and tetrachloroethylene for the ability to decrease the methylation of DNA. We will determine the mechanism for the decreased methylation of DNA. We also 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 methylene chloride administered by inhalation decreased the methylation of the c-myc gene in the lungs, livers, kidneys, and bladders of mice. Thus, it was demonstrated that administering the carcinogens by inhalation causes hypomethylation in the lungs 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; it allows the determination of the ability of the non-carcinogens carcinogens 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. Therefore, another procedure is being used that allows us to determine the methylation of a gene, and as long as its sequence is known, the gene will be amplified by a 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, 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 dichloracetylene (DCA) or trichloroacetic acid (TCA). In a limited number of samples, both dimethylarsinic acid and sodium arsenite appeared to decrease the methylation of the IGF-2 gene. However, because IGF-2 is imprinted, the two chromatids containing the gene have different patterns of methylation. This has hampered the interpretation of our results. This was not a problem when we investigated tumors; in tumors, both chromatids have a very low level of methylation. Therefore, we are attempting to use a different gene, the tumor suppressor gene p21Cip1/Waf1/Sdi1 (p21).
In addition, we are attempting to develop another procedure using an antibody 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.
DNA methylation is one of the mechanisms that controls transcription. DNA methylation also is involved in chromosomal stability. Thus, DNA hypomethylation induced by non-genotoxic 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 non-genotoxic carcinogens can cause cancer. Therefore, we are attempting to determine whether non-genotoxic carcinogens not only decrease the methylation of the p21 gene outside of its promoter, but also increase the methylation inside its promoter.
We will determine the mechanism for the decreased methylation of protooncogenes. 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; therefore, DCA and TCA would have to increase cell proliferation and DNA replication for hemimethylated sites to require 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 appears to result from blockage of DNA methyltransferase (DNA MTase) to the hemimethylated sites in DNA. This is the mechanism for non-genotoxic carcinogens that we are investigating.
This study is to determine whether the prevention of DNA methylation is the result of decreased polyamine levels or increased methylation of histones. These possible mechanisms were chosen because polyamine synthesis and histone methylation are dependent on SAM, similar to DNA methylation.
We 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. The aim also is to determine whether tumors induced by arsenic compounds have molecular alterations consistent with arsenic induced general DNA hypomethylation and hypermethylation of the promoter of tumor suppressor genes.
We started a study to determine whether methionine prevention of DNA hypomethylation induced by DCA resulted in prevention of DCA-promotion of N-methyl-N-nitrosourea (MNU)-initiated liver tumors in mice. However, the MNU induced lymphomas that occurred early in the study. Although we found MNU-induced lymphomas in previous studies, their incidence was less than 5 percent and occurred at approximately one year. In this study, the lymphomas were present by 2 months; the study was terminated. We have started a new study in which we will not initiate the mice with MNU, but rather, we will determine the effect of methionine on DCA-induced liver tumors.
We also have started a study to induce lung tumors in mice with dimethylarsinic acid and sodium arsenite. This study will supply lung tumors to determine whether hypermethylation of tumor suppressor genes is involved in arsenic carcinogenic activity. We will contrast the molecular biology of arsenic-induced lung tumors to that of lung tumors induced by genotoxic carcinogens, benzo(a)pyrene, and vinyl carbamate. We have the benzo(a)pyrene and vinyl carbamate induced lung tumors from our prior studies. Thus, this study will determine whether arsenic induces lung tumors by a mechanism similar to genotoxic carcinogens.
The expected results of this project should provide a better understanding of the mechanism of non-genotoxic carcinogens, specifically the involvement of increased DNA hypomethylation and the hypermethylation of tumor suppressor genes. This will demonstrate that an epigenetic mechanism is involved in the carcinogenic activity of these carcinogens. Using the ability to induce DNA hypomethylation as a biomarker, the dose-response relationship of the carcinogens could 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 include the determination of the ability of sodium arsenite and dimethylarsinic acid to alter the methylation of the DNA and the p21 gene. The other carcinogen we have so far to evaluate is arsenic; its metabolite might cause hypomethylation by decreasing the availability of SAM or inhibiting 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 through which the carcinogens block the accessibility of DNA MTase to CpG sites, and thus prevent methylation, is being investigated. We will determine whether this blockage is due to decreased polyamine synthesis or methylation of histone.
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 DCA-induced cancer. We will be investigating the specificity of the effect of methionine on DNA methylation by determining whether it prevents DCA enhancement of cell proliferation, glycogen accumulation, oxidative damage, and peroxisome proliferation.
We plan to determine whether arsenic induced lung tumors in mice possess DNA hypomethylation, hypermethylation of the promoter of p21, and other tumor suppressor genes. We also will determine whether the molecular biology of arsenic-induced lung tumors is similar to that of the genotoxic carcinogens, benzo(a)pyrene, and vinyl carbamate.
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.||