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. Recently, using thisimproved comet assay protocol for evaluating single strand break repair capacity (SSB-RC) in unstimulated cryopreserved human peripheral blood mononuclear cells (PBMCs). This methodology facilitates control of inter-experimental variability (Trzeciak et al, 2008). The fast component of SSB repair (F-SSB-RC) was assessed using a novel parameter, the initial rate of DNA repair, and the widely-used half-time of DNA repair. The slow component of SSB repair (S-SSB-RC) was estimated using the residual DNA damage after 60 min. We have examined repair of γ-radiation-induced DNA damage in PBMCs from four age matched groups of male and female whites and African-Americans between ages 30-64. There is an increase in F-SSB-RC with age in white females (p<0.01) and non-significant decrease in FSSB-RC in African-American females (p=0.061). F-SSB-RC is lower in white females than in white males (p<0.01). There is a decrease in F-SSB-RC with age in African-American females as compared to white females (p<0.002) and African-American males (non-significant, p=0.059). Age, sex and race had a similar effect on intercellular variability of DNA damage in γ-irradiated and repairing PBMCs. Our findings suggest that age, sex and race influence SSB-RC as measured by the alkaline comet assay. SSB-RC may be a useful clinical biomarker. Currently, there is no universally accepted panel of useful markers of oxidative stress in humans even though many lines of evidence suggest that oxidative DNA damage and other forms of oxidative stress influence the development of age-associated disease. This study examines various markers of oxidative DNA damage, oxidative stress and inflammation including: the baseline level of single strand breaks (SSBs) and oxidative base lesions recognized by DNA glycosylases (Endo III and Fpg) in peripheral blood mononuclear cells (PBMCs), red blood cell glutathione concentration (RBC GSH), hemoglobin degradation products, plasma protein carbonyls, and serum levels of alpha-tocopherol and high-sensitivity C-reactive protein (hs-CRP). In addition, we examined SSB repair capacity (SSB-RC). We studied four age-matched 24-individual groups of male and female whites and African-Americans aged 30-64 years. We found that females have higher SSB levels than males (p=0.013). We observed a significant negative correlation between SSB-RC and SSB level (p=0.041). In addition, we found a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and hs-CRP (p=0.022). Higher levels of residual DNA damage were associated with increasing concentration of hs-CRP in males as compared to females (p=0.002). RBC GSH concentration was found to positively correlate with the levels of Endo III-sensitive sites (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). Our results suggest a complex relationship between measures of oxidative stress and clinical parameters used frequently and believed to reflect inflammation or oxidative stress. Further investigations should target specific forms of oxidative stress to uncover the relevance of specific forms of oxidative damage, stress and inflammation to clinical measures and disease risk.
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