Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.

Acute hypoxia and reoxygenation-induced DNA oxidation in human mononuclear blood cells.

Research indicates that exposure to hypoxia is associated with oxidative stress. In this investigation, healthy subjects were exposed to hypoxia by inhalation of 10% oxygen for 2h (corresponding to 5500m above sea level). The levels of strand breaks and oxidatively damaged purine bases, measured by the comet assay, and the expression of genes involved in DNA repair of oxidatively damaged DNA were investigated in mononuclear blood cells (MNBC) at baseline, after 2h of hypoxia, 2h of reoxygenation, and 1 day and 8 days after the exposure. The level of strand breaks and oxidized purine bases in MNBC increased following both the 2h of hypoxia and the 2h reoxygenation period, whereas this effect was not observed in unexposed subjects. The expressions of oxoguanine DNA glycosylase 1 (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), nei endonuclease VIII-like 1 (NEIL1), and mutY homolog (MUTYH) were unaltered throughout the experiment in both groups of subjects, indicating that DNA repair genes are not up-regulated by the hypoxia and reoxygenation treatment. Taken together, this report shows that inhalation of 10% oxygen for 2h is associated with increased number of oxidized DNA lesions in MNBC, but acute hypoxia may not inflict upon the regulation of genes involved in repair of oxidized DNA.

Pain and quality of life for patients with venous leg ulcers: proof of concept of the efficacy of Biatain-Ibu, a new pain reducing wound dressing.

Wound pain is a serious problem for elderly patients suffering from chronic leg ulcers, and it may lead to reduced wound healing rates and reduced quality of life. Biatain-Ibu Non-adhesive (Coloplast A/S), a new pain-reducing moist wound healing dressing containing ibuprofen was tested for pain reduction, safety, and efficacy on 10+2 patients in a single-blinded crossover study against Biatain Non-adhesive (Coloplast A/S). Pain was measured with a Numeric Box Scale before, during, and after dressing change. Quality of life was measured using the World Health Organization-5 Well-Being Index. Dressing moist wound healing properties such as absorption capacity and leakage were tested together with assessment of wound exudate and blood plasma content of ibuprofen. Use of the Biatain-Ibu foam dressing correlated with a decrease in pain intensity scores from 7 in the run-in period to approximately 2.5 in the Biatain-Ibu treatment phase. Quality of life measures were improved which together with the reduced pain could contribute to faster wound healing. The moist wound healing properties of Biatain-Ibu were similar to that of the Biatain Non-adhesive and ulcer size was reduced by 24% during the treatment period. Neither side effects nor systemic plasma concentrations of ibuprofen were observed. These data indicate that Biatain-Ibu could reduce persistent and temporary wound pain, increase Quality of life, was found safe to use, and had excellent moist wound healing properties.

Intra-laboratory comet assay sample scoring exercise for determination of formamidopyrimidine DNA glycosylase sites in human mononuclear blood cell DNA.

Oxidative DNA damage detected by the comet assay as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, almost as a rule is reported as comet assay score rather than numerical sites in the genome, probably because the latter requires X-ray calibration. We compared the ability of five experienced and five inexperienced comet assay investigators to detect a dose-response relationship in irradiated A549 lung epithelial cell culture samples (0, 10 Gy and three samples of 5 Gy), based on an arbitrary five class scoring system. The samples were scored on three different occasions, thus allowing determination of the variation in sample scoring. All investigators qualitatively distinguished between samples in a dose-dependent manner, albeit with large variation in the slope and intercept of dose-response curves. There was a tendency that investigators with experience in scoring A549 cells had more consistent results than experienced investigators who had only scored lymphocytes or inexperienced investigators. The inexperienced investigators improved their scoring ability during the three sessions. Subsequently we showed that the variation in baseline level of FPG modifications in mononuclear blood cells of five healthy humans was lower when investigators used their individual X-ray calibration curve as compared to a common calibration curve. In conclusion, this study showed that comet assay investigators score differently when using a five class scoring system, which indicates that more consistent estimations of FPG sites in the genome are obtained by use of investigators' individual X-ray calibrations.