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ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY
Tennant, A H., G W. Knapp, AND A D. Kligerman. ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY. Presented at GEMS fall meeting, Chapel Hill, NC, 11-05-03.
ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY, Alan H. Tennant1, Geremy W. Knapp1 and Andrew D. Kligerman1, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
The use of in vitro cellular models may aid in the development of biomarkers that can potentially indicate differential sensitivities to DNA damage and repair efficiencies in children and adults. To assess the potential differences in the repair capabilities of cells of younger and older individuals, the alkaline single cell gel assay can be used to measure both specific DNA damage caused by alkylation and its subsequent repair. Human skin fibroblasts from healthy males ten years old and less than one year old were obtained from Coriell Cell Repository and were cultured in minimal essential medium (MEM) supplemented with 15% fetal calf serum. Cells were exposed to the alkylating agent methyl methanesulphonate (MMS) for 1 hour in MEM supplemented with 0.15% fetal calf serum at concentrations of 0, 25, 50 and 100 uM. Following exposure, cells were washed and detached with 0.25% trypsin into a single cell suspension. The alkaline single cell gel assay was performed following the methods of Tice et al. (1991). Cells were analyzed using fluorescence microscopy and the software package Komet 5.0 (Kinetic Imaging). DNA damage as indicated by tail moment increased in a concentration dependent manner among all samples, and there were no significant differences in damage between cells from younger and older individuals. Further studies will measure decreases in DNA damage over time following exposure to MMS as an indicator of DNA repair in these cells.
[This abstract does not necessarily reflect EPA policy.]