Arsenic is a human carcinogen found at high concentrations in the drinking water of millions of people world wide. The primary target organ for ingested arsenic is the skin, and cancer also develops in the bladder, kidney and liver. Although is not a mutagen, the irreversible nature of arsenic exposure suggests that this carcinogen produces heritable alterations in DNA. The hypothesis for this proposal is that arsenic exerts two separate effects that synergize to produce permanent, non-mutational changes in DNA: stimulation of DNA replication and perturbation of methyl metabolism. In this model, the end result is altered gene methylation, producing some cells with deregulated expression of genes critical to transformation. The hypothesis is supported by the fact that arsenic is extensively methylated during metabolism and has the ability to activate the signal transduction pathway. Using primary human fibroblasts, the specific aims are: 1) To determine the cellular mechanisms by which acute arsenic exposure sensitizes cells to subsequent mitogenic stimulation by analyzing cell cycle kinetics, proliferation and activity of the mitogenic and stress signaling pathways; 2) To determine the magnitude and concentration of dependence for arsenic to interfere with DNA methylation, either by inhibiting DNA methyltransferase or by disrupting methyl metabolism; 3) to determine the effects of chronic arsenic exposure on characteristics associated with proliferation, i.e., phenotypic transformation and the cellular responses to mitogens and stressors; 4) To determine if chronic arsenic exposure produces sustained changes in DNA methylation, both overall genomic methylation and the methylation of specific genes involved in cancer. This study is unique in combining two mechanisms, altered proliferation and disrupted methylation, into a unified hypothesis for arsenic carcinogenicity. The results of the experiments proposed herein will provide and a better understanding of the complex actions of arsenic and can be used to minimize the impact of arsenic exposure on human health and in formulating exposure guidelines.