DNA REPAIR AND SUSCEPTIBILITY TO ENVIRONMENTAL AGENTS

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DESCRIPTION (provided by applicant) DNA damage is recognized as an important mode of action in carcinogenesis. Exposure to environmental agents may result in oxidative and alkylation damage leading to formation of a number of mutagenic DNA adducts. Cells can respond to DNA damage by coordinated induction of DNA repair and it has been shown that differences in the accumulation and removal of DNA lesions in different organs and in various species exist. Therefore, the investigators will test the hypothesis that the extent of induction of DNA repair in response to oxidative and alkylation damage varies in different species and tissues, and that such variation contributes to species and site-specific mutagenic and cytotoxic effects of environmental chemicals. To obtain data relevant for human risk assessment and to explore mechanisms of DNA repair, the experiments will be carried out using cultured cells isolated from various human tissues (e.g., liver, brain, skin, bone marrow) and matching tissues from mouse. First, the investigators will evaluate the basal levels of expression of a cluster of nearly 60 DNA repair genes involved in base excision, nucleotide excision and mismatch repair pathways in human and mouse cells. Expression of DNA repair genes will be studied on the mRNA and protein level by using multiprobe RNase protection assay and Western blotting, respectively. Second, they will compare the transcriptional response of DNA repair genes to oxidative and alkylation damage. Human and mouse cells will be exposed to ionizing radiation, tetrachlorohydroquinone, methyl methanesulfonate, or N-methyl-Nnitrosourea. Expression of DNA repair enzymes will be compared between cell types and species using cDNA arrays. Protein levels will be verified by Western blotting, or by proteomics analysis. Third, the investigators will determine whether changes in the activity of base excision, nucleotide excision and mismatch repair caused by environmental agents correlate with a degree of induction of DNA repair genes. Here, the efficiency of cell-free extracts to recognize and repair lesions in vitro, as well as a number of basic sites and single strand breaks will be compared. The investigators expect that the degree of induction of DNA repair genes as well as the activity of repair will differ between cells and species and that unique patterns of response to each treatment can be identified. DNA repair pathways have been shown to interact with p53-mediated signaling to apoptosis; therefore, the final aim is to determine the role of p53 in tissue-specific regulation of transcriptional responses of DNA repair genes to oxidative and alkylation damage by environmental agents. Here, the investigators will use cells isolated from wild type and p53-null mice and treat them in vitro as detailed above. Accumulation of DNA lesions and changes in expression of DNA repair genes will be studied. The investigators anticipate that p53-dependent and independent pathways involved in DNA repair following environmental exposure will be identified. Collectively, these studies will compile a matrix of data on how expression and activity of a cluster of genes involved in DNA repair varies among different organs from both humans and mice. These experiments will also provide crucial mechanistic insights into possible differences in species- and tissue-specific responses to DNA damage induced by environmental agents.