Arsenic exposure via drinking water is associated with increased risk of human cancers, as well as a number of other dysfunctions. The investigators have demonstrated that although arsenite (the likely carcinogenic form of arsenic) is not a gene mutagen, it acts as a comutagen by interfering with DNA repair. However, DNA repair enzymes in vitro are not sensitive to arsenite implying that arsenite s effect on DNA repair in cells is not via enzyme inhibition. The investigators suggest that inhibition of DNA repair by arsenite results from an interference with p53-dependent pathways controlling cellular responses to DNA damage. Human exposure to arsenic, e.g. in drinking water, occurs over a long period of time. This proposal will address the effects of long term, low level arsenite treatment of a number of p53-related endpoints. Because they have found that rodent cells exposed to low levels of arsenite develop tolerance, whereas human cells do not, the emphasis will be on human cells.
The specific aims are first to determine the ability of arsenite and its metabolite dimethylarsinic acid (DMAA) to interfere with the cellular resoponse to x-ray-induced DNA damage in normal human diploid fibroblasts and keratinocytes. DNA-damage-in-ducible responses include transient cell cycle arrest, increase in p53 protein abundance, and the up-regulation of genes downstream of p-53(particularly gadd45 and p21). Second, the investigators will determine whether human cells which have undergone mutation, gene amplification, or transformation as a result of long term, low level arsenite or DMAA exposure(alone or with low doses of UVB) show increased frequencies of genomic instability compared with similarly treated cells which were not altered. Third, they will study the role of oxidant stress in arsenite's action by determining whether any effects seen in aims 1 (where p53-dependent fucntions are measured) and 2 (where genetic effects are measured) can be blocked by ascorbate, atocopherol, or N-acetyl-cysteine. Lastly, by expression cloning for genes which confer resistance to arsenite, they have cloned 2 cDNAs, fau (a purported tumor suppressor gene) and asr2 (whose product plays a role in apoptosis). The investigators will determine whether fau or asr2 will, when overexpressed, contribute to genomic instability in human cells.
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