Prompt and delayed nonsteroidal anti-inflammatory drug-photoinduced DNA damage in peripheral blood mononuclear cells measured with the comet assays.

Nonsteroidal anti-inflammatory drug (NSAID)-photoinduced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid-photoinduced DNA damage was promptly induced (i.e. observed at relatively low radiation doses), Ketoprofen-photoinduced DNA damage was delayed. This prompt and delayed effect is observed with UVA (320-400 nm), UVB (290-320 nm) and solar-simulated radiation and is attributed to the different photochemical properties of NSAID. The results from these experiments, carried out in living cells, confirm the speculations of NSAID-photoinduced DNA damage brought up by the many experiments conducted in solution.

The effect of the ratio of CD4+ to CD8+ T-cells on radiation-induced apoptosis in human lymphocyte subpopulations.

PURPOSE: Apoptosis occurs spontaneously in cultured human peripheral blood lymphocytes but is enhanced by exposure to ionizing radiation. Subpopulations of lymphocytes are known to have varying radiosensitivities to radiation-induced apoptosis. The purpose of this study was to examine the radiation-induced apoptotic response of CD4(+) and CD8(+) T-cells incubated as a complete lymphocyte population. MATERIALS AND METHODS: Using a four-colour flow-cytometry method, which measures annexin-V binding to phosphatidyl serine and propidium iodide, spontaneous and radiation-induced apoptosis was measured in the total lymphocyte fraction and in CD4(+) and CD8(+) T-cell subpopulations. RESULTS: It was found that CD8(+) T-cells were more sensitive to radiation-induced apoptosis than CD4(+) T-cells at doses up to 2 Gy. The yield of radiation-induced apoptosis in the total lymphocyte fraction decreased with increasing ratios of CD4(+) to CD8(+) T-cells (CD4/CD8 ratio). By manipulating the CD4/CD8 ratio within lymphocyte cultures, it was found that the CD4/CD8 ratio had a dramatic effect on the yield of spontaneous apoptosis of total lymphocytes fraction and CD4(+) T-cells but not CD8(+) T-cells. CONCLUSION: The CD4/CD8 ratio affects the apoptotic response of human lymphocytes and CD4(+) T-cells.

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay.

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.

DNA damage and apoptosis in the immature mouse cerebellum after acute exposure to a 1 mT, 60 Hz magnetic field.

Several recent studies have reported that whole-body exposure of rodents to power frequency magnetic fields (MFs) can result in DNA single- and double-strand breaks in the brains of these animals. The current study was undertaken to investigate whether an acute 2h exposure of a 1 mT, 60 Hz MF could elicit DNA damage, and subsequently apoptosis, in the brains of immature (10-day-old) mice. DNA damage was quantitated at 0, 2, 4, and 24h after exposure using the alkaline comet assay. Apoptosis was quantitated in the external granule cell layer (EGCL) of the immature mouse cerebellum at 0 and 24h after exposure to MF by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. While increased DNA damage was detected by tail ratio at 2h after MF exposure, no supporting evidence of increased DNA damage was detected by the other parameters. In addition, no similar differences were observed using these parameters at any of the other post-exposure times. No increase in apoptosis was observed in the EGCL of MF-exposed mice, when compared to sham mice. Taken together, these results do not support the hypothesis that acute MF exposure causes DNA damage in the cerebellums of immature mice.

DNA damage detection technique applying time-resolved fluorescence measurements.

A novel DNA damage detection technique based on the characteristic fluorescence lifetimes exhibited by Pico-Green-single-stranded DNA and -double-stranded DNA complexes is employed to establish the damage produced on DNA isolated from sheep white blood cells following gamma radiation. This technique, which incorporates key concepts such as alkaline unwinding buffers and higher unwinding rates of damaged DNA compared to undamaged DNA, allows for the differentiation of DNA damage resulting from doses of gamma radiation in the 0-100-Gy range, with the potential of analyzing samples consisting of as little as 10(4) cells. Experiments were carried out using commercial DNA sources as well as DNA isolated from sheep white blood cells, suggesting its potential for use with isolated DNA from virtually any eukaryotic cell.