The objective of this application is to further characterize the molecular and biological events that occur following genotoxin exposure of cells and tissues derived from human fetal brain (glial and neuronal cells), kidney (proximal tubular and glomerular epithelial cells), and skin (fibroblastic and epithelial cells). DNA damage and repair will be characterized and quantitated as a function of DNA modification, organ and cell type as related to individual, organ and cell sensitivity. UV radiation and nitrosoureas will be used to represent direct acting genotoxins which induce well characterized bulky (pyrimidine dimers) and non-bulky (N7 and 06-alkylguaine, 04 and 02-alkylthymine, N3-alkyladenine, phhosphotriester, and apurinic sites) lesions in the DNA. Studies will be done, when possible, simultaneously with both fresh human fetal tissue and cultured cells derived from the same specimen. The inhibition, saturability and inducibility of the repair enzymes with related and unrelated agents either separately or in combination with different agents (i.e., UV, alkylator, AAAF) will be investigated in cell culture as a function of dose and time of exposure to genotoxin and in specific stages of the cell cycle, e.g. G1 vs S phase. Indiviudal cell and organ sensitivity will be measured by the dose and time dependent changes in cell morphology and growth rate, cytotoxicity, anchorage independent growth and nucleic acid and protein synthesis. The studies of this renewal research grant are designed to provide new information and insights into the mechanisms of genotoxicity on cellular function in relation to human organ and cell type. The long term goal of this research program is to interrelate the molecular events of genotoxic DNA damage and DNA repair of specific DNA modifications to the biological parameters in humans in vivo and in vitro.
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