DNA damage and repair measurements from cryopreserved lymphocytes without cell culture--a reproducible assay for intervention studies.

摘要

Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When gamma-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after gamma-irradiation exposure, and DRC--measured as tail moment--were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H(2)O(2) was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37 degrees C (CVs. ranging from 8 to 35%) than for the more standard 4 degrees C protocol. Analyzing moment arm--the average distance of DNA migration within the tail--yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation.