The clinical applications of the COMET Assay are varied ranging from assessment of chemotherapy agent geno- and cytotoxicity to correlation of oxidative DNA damage levels in spermatozoa with male infertility. Use of the Alkaline Comet Assay to assess DNA repair capacity (DRC) in human population studies has been limited by difficulties in controlling for inter-experimental variability, developing appropriate internal standards and establishing a methodology for cryopreserved lymphocytes appropriate for use in this assay.
The aim of the work is to develop an accurate, reproducible and efficient comet assay methodology for evaluating DNA repair capacity (DRC) as part of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Here we show the preliminary work on assessing repair of ?-radiation-induced DNA damage in a small cohort from this study. Our work thus far shows that unstimulated human cryopreserved lymphocytes can be used to measure DRC using the comet assay. Furthermore, we have successfully applied standardization of data obtained in separate experiments. Furthermore, we have successfully applied standardization of data obtained in separate experiments. Our biomonitoring study includes data obtained for lymphocytes derived from four age-matched groups of female Caucasians, female African Americans, male Caucasians, and male African Americans. We found that the DNA repair capacity of ?-radiation-induced single strand breaks and AP sites is decreased in Caucasian females, as compared to males and African-American women as assessed by the alkaline comet assay. This decrease in DNA repair capacity in Caucasian females is associated with the presence of a small population of lymphocytes having higher than average levels of DNA damage after ?-irradiation at 30-and 60-min repair time points. The preliminary data presented here suggests that DRC does not decline with age in the small selected cohort. These preliminary findings will be compared with DNA damage and repair data from others in the cohort and correlated with the multidisciplinary variables available in this study which include co-morbid illness, tobacco use, alcohol and drug abuse, occupational exposure, and clinical parameters. The DNA repair capacity (DRC) in the cells may depend on a variety of factors. Wei et al. has shown that females, persons with a family history of cancer and patients with active cancer exhibit the lowest DRC as assessed by the host cell reactivation assay. There is no agreement on the influence of smoking on DRC; different report show both increases and decreases in DRC among smokers. Despite many suppositions in the literature, there is no clear correlation between age and DRC. Some reports indicate that DNA repair declines with age while others find no effect of age on DRC in healthy individuals. The Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study is a community-based, epidemiologically driven multidisciplinary research effort designed to focus on evaluating health disparities in socioeconomically diverse Caucasians and African Americans in Baltimore. One of the domains of the HANDLS study examines the possible role of oxidative stress and defects in DNA repair in the development of age associated disease.
The aim of this work is to develop an accurate, reproducible and efficient comet assay methodology for evaluating DNA repair in cryopreserved lymphocytes obtained as part of the HANDLS study. We show first that cryopreserved lymphocytes can be used in DNA repair studies performed using comet assay. We decided to use cryopreserved lymphocytes since blood will be drawn only once from each individual, blood samples from several individuals will be obtained on the same day and using fresh lymphocytes does not permit multiple independent analyses of DNA repair capacity. Unfortunately, all available reports showed that DNA repair capacity could not be measured using the comet assay in unstimulated cryopreserved lymphocytes. We have developed a protocol for lymphocyte cryopreservation and the comet assay that permits us to study DNA repair in unstimulated cryopreserved lymphocytes. Analysis of DNA repair in lymphocytes from multiple individuals requires many comet assay experiments to be done; however, inter-experimental variability between the experiments must be addressed. To resolve this issue, we have applied standardization techniques for the analysis of DNA repair data for each experiment utilizing negative and positive internal standards.
| Jacob, Kimberly D; Noren Hooten, Nicole; Trzeciak, Andrzej R et al. (2013) Markers of oxidant stress that are clinically relevant in aging and age-related disease. Mech Ageing Dev 134:139-57 |
