The objective of this grant proposal is to further characterize the way in which human fetal cells respond to genotoxic agents. Studies will quantitate and compare, in cells derived from human fetal brain, dermis and kidney: the levels of initial DNA damage, repair, replication, binding and DNA repair activities. These cells were chosen because: human epidemiological data indicates that the nervous system and kidney exhibit high levels of cancer in the newborn, and our previous studies indicate that these human fetal cells exhibit the most significant differences in response to genotoxic agents. Studies are designed so as to further characterize these differences by correlating repair synthesis to replication potential to adduct persistence to DNA repair enzymatic activities. Genotoxin agents of three different types (ultraviolet radiation, dimethyl and diethyl sulfate, and methyl and ethyl nitrosourea) will be used to produce different types (thymidine dimers, N7-alkylguanine, N3-alkyladenine and 06-alkylguanine) and extents of modification in cellular DNA. Persistence of each modification will be quantitated by N-glycosylase/endonuclease specific for thymidine dimers and HPLC methods for separating alkylated bases. Using specifically radiolabeled DNA substrates the level of N-glycosylase, apurinic endonuclease and alkyltransferase will be determined and compared in each cell type. We have a unique resource of human fetal tissue and novel culturing techniques. These studies should thus further characterize how human fetal cells respond to genotoxic compounds during human fetal development.
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