The alkaline comet assay is a sensitive and relatively inexpensive technique used for the detection of DNA damage. One of the major advantages of the alkaline comet assay is the possibility of analysis of DNA damage and repair in single cells. Furthermore, a small number of cells is required for this assay which is particularly advantageous for population analysesCells obtained from participants are suspended in low melting point agarose (LMP) and placed on a slide. Subsequently, baseline or induced DNA damage levels and/or DNA repair capacity are assessed after alkaline lysis, alkaline unwinding and sample electrophoresis. Levels of DNA damage and repair are assessed by analyzing the morphology of cells using fluorescence microsopy. ? Most of the alkaline comet assay protocols used in biomonitoring employ peripheral blood mononuclear cells (PBMCs) that facilitate wide application of the comet assay since obtaining blood from human subjects is relatively easily and not particularly invasive. However, the use of the PBMCs in epidemiologic studies of DRC is still challenging due to the technical and logistical difficulties in utilizing fresh lymphocytes and the lack a validated protocol for use with cryopreserved PBMCs. Furthermore, the relatively high inter-experimental variability existing in the comet assay presents some difficulty in detecting inter-individual differences in response to genotoxic factors. ? ? We have developed, and implemented modifications to the alkaline comet assay which may increase ability of the assay to detect subtle differences in DRC.We have now shown that cryopreserved lymphocytes can be used in DNA repair studies performed using comet assay. Irradiated PBMCs removed DNA damage, whereas unexposed PBMCs did not accumulate DNA lesions during 2-h incubation after exposure to ionizing radiation. We examined the dose-effect relationship between -radiation and the DNA damage level in fresh and cryopreserved AG10097 cells, since we planned to use these cells as internal standards in the HANDLS DNA repair study. Cells embedded in agarose were exposed to -radiation at the dose range 0-10 Gy. There was no difference in the average level of DNA damage measured by any of the three traditional comet assay parameters between fresh and cryopreserved AG10097 cells as analyzed by two-way ANOVA (p > 0.05). Furthermore, similarities in the histogram distribution patterns between fresh and cryopreserved AG10097 cells were observed for each comet assay parameter. Thus, cell cryopreservation did not alter the induction of DNA damage by -radiation. We also analyzed Olive Tail Moment (OTM), tail DNA and tail length for linearity in the quantification of the amount of -radiation-induced DNA damage in cells. The OTM data shows linearity for radiation doses 0-6 Gy with moderate saturation occurring in the range of 6-10 Gy. The tail DNA data shows slight saturation-related departure from linearity in the dose range 0-6 Gy and moderate departure at higher studied doses. The tail length values increase rapidly between 0-2 Gy and then reach plateau. The saturation of this parameter likely occurs at a -radiation dose less than 2 Gy. The maximum dose at which no significant departure from linearity for these comet assay parameters appears is estimated at approximately 6 Gy. We also address the problem of inter-experimental variation in the comet assay. 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. We have developed a protocol for estimation of the rate of fast and slow component of DNA repair in human population samples utilizing unstimulated cryopreserved PBMCs and data standardization. We have also introduced initial rate of DNA repair, a new DNA repair parameter showing the rate of DNA repair immediately after a genotoxic exposure. We found that DRC in cryopreserved PBMCs was comparable to that of fresh PBMCs suggesting that cryopreserved PBMCs can be used in retrospective DRC analysis in human population studies. Thus, our refinement of alkaline comet assay procedure will facilitate a more detailed analysis of DNA repair in human population using cryopreserved PBMCs.
| 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 |
