Generally this methylation occurs at cytosines that are containedin a symmetrical CpG nucleotide sequence (24). Alterationsin global levels and regional changes in the patterns ofDNA methylation are commonly observed in the early stages ofneoplasia (15,33). Errors in DNA methylation begin to appearearly in tumor development, prior to the appearance of overtneoplasia (34,35). In particular, it is thought that aberrant DNAmethylation could be important in the development of livercancers (36) and is a possible epigenetic mechanism that underliesthe aberrant expression of genes involved in the mouse livercarcinogenesis (16,37). This fortifies the concept that the hepaticDNA methylation errors observed in the present study afterarsenic exposure may have implications in tumor causation. Theliver is clearly a target of arsenic carcinogenesis in humans (2),and proliferative liver lesions occur after inorganic arsenicexposure in mice ranging from hyperplasia to hepatocellularcarcinoma (5,7,8). It is also of interest that methyl-deficientdiets, which produce DNA hypomethylation and steatosis, also produce hepatocellular tumors (28,29). This is consistent with the present results where DNA methylation changes are associated with fatty changes in the liver. It is unclear if these hepatic changes would have resulted in eventual tumor formation, but this may have been the case had the exposure period been extended. However, the present results clearly indicate chronic arsenic exposure in vivo can cause generalized DNA methylation errors that could lead to liver tumor formation. DNA hypomethylation can be induced by methyl-deficient diets or chemically induced methyl donor group depletion, including chronic depletion of the cellular SAM pool (29). Such depletion is thought to induce methylation errors by causing insufficient methyl groups for DNA methylation during replication (37,38). Indeed, feeding of methionine and choline-deficient (i.e. `methyl-deficient’) diets results in hepatocellular tumor formation in mice associated with SAM depletion and DNA hypomethylation (28,29,37). In fact, the combination of methyl-deficient diets and arsenic produced DNA hypomethylation in mice (38), although in this study arsenic alone was ineffective. It is noteworthy that steatosis associated with feeding methyl-deficient diets in this study was made more severe by arsenic exposure (38). In rats fed a choline-devoid diet for up to 14 months, hepatic lesions develop and progressed through two distinct stages, the first characterized by severe steatosis and an increase in cell turnover and the second by fibrosis and the development of hepatocellular carcinomas (39). Therefore, steatosis seen in this study may well be related to DNA hypomethylation, which in turn, may be a precursor of liver neoplasia. Chronic methyl depletion is probably not the only mechanism by which arsenic induces hypomethylation and factors such as direct inhibition of DNA methyltransferase (9) probably also contribute to this response.
Hypomethylation in the promoter region of various genes can control their expression via control of transcription (24,25). The presence of methylated CpG islands in promoter regions typically suppresses expression, while hypomethylation leads to over-expression (24). This process is thought to be due to the presence of 5-methylcytosine that interferes with the binding of transcription factors or other DNA-binding proteins thereby blocking transcription. In different types of tumors, aberrant methylation of CpG islands in the promoter region has been observed for many cancer-related genes, such as c-myc, and Ha-ras(38,40). Decreases in the 5-methylcytosine content of DNA and the hypomethylation of individual genes are common early events found in many human and animal tumors (29,34,35,41). In the present study, when the genomic sequencing technique to map methylation of the 13 CpG rich sites within ER-a promoter region, they all showed much less methylation after arsenic exposure. Overall, there was almost a 90%decrease in the methylation of the ER-a promoter region after arsenic exposure. These results clearly indicated that DNA hypomethylation of ER-a gene occurred after chronic arsenic exposure, and thus was potentially responsible for Era over-expression, since several studies indicate ER expression is inversely associated with methylation (26,27). Thus, chronic arsenic exposure can introduce DNA methylation errors that may activate critical genes in oncogenesis, at least in the liver.