One of the prices we pay for living in an industrial and technological society is exposure to a variety of chemical and radiation mutagens. Examples of this exposure are contaminating levels of Cs-137 remaining from the Chernobyl disaster and increased exposure to UV due to thinning of the ozone layer. Both events are expected to result in increased numbers of cancers. Although it is possible to readily detect this exposure if it occurs at high dose, it is far more difficult to detect this exposure if it occurs at low dose or if the possibility of exposure is raised after it has occurred. One potential means of addressing this problem is to determine the spectrum of mutations induced by a specific mutagen and then determine if this spectrum is present in genomic DNA in cells taken from individuals at risk. This grant proposal involves using a cultured mouse stem cell system to determine more accurately the spectrum of mutation induced by ionizing radiation. The first specific aim is to determine the mutational spectrum induced by high dose high and low LET ionizing radiation and compare and contrast this spectrum with those spectra produced by equally genotoxic doses of UV and an alkylating chemical mutagen, as well as with the spontaneous spectrum. Having defined the ionizing radiation spectrum, the second specific aim is to determine the lowest possible dose at which all or part of this spectrum can be recognized. The third and final specific aim is to determine if the ionizing radiation induced mutational spectrum is altered in morphologically differentiated cells derived from the stem cells. Such a comparison has not been performed previously. The mouse cell lines to be used are unique because they have heterozygous deficiencies for the purine salvage gene aprt (adenine phosphoribosyltransferase) and because they contain two chromosome 8 homologs, which harbor the aprt gene, that are distinguishable with a molecular analysis. Cells with homozygous deficiencies, which will be induced with the above mutagens, will be selected and a molecular analysis used to elucidate and enumerate the spectrum of mutations. This molecular analysis will include Southern blots, PCR, and DNA sequencing.
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