Diversity of peripheral blood mononuclear cells as revealed by a novel multiple microgel "comet assay".

Multiple microgel comet assay (MMCA) is a metho-dological adaptation of the single-cell gel electrophoresis assay in which we have introduced the use of standard agarose plug molds in an attempt to improve and expand the applications of the assay. We focused on the study of the heterogeneity of peripheral blood mononuclear cells (PBMC) at the level of the basal single-strand breakage and the DNA damage induction caused by ionizing radiation. Differences among subpopulations were also investigated at the level of chromatin organization and methylation after NotI digestion of microgel-embedded cells. In parallel experiments, the NotI-digested nucleoids were also analyzed with the use of pulsed-field gel electrophoresis (PFGE) and the DNA migration patterns were compared with the corresponding patterns from the MMCA. Significant heterogeneity in the distribution of the oxidative DNA damage, as well as intracellular variations in the NotI digestion patterns were observed in the cell population of PBMC. The combined use of both the comet assay and PFGE provides a useful model for analysis of variation in DNA damage in individual cells as well as information on size of DNA fragments.

Effects of radical scavengers on radiation-induced DNA double strand breaks.

PURPOSE: To investigate possible effects of Tris and phenol on the dynamic properties of gamma-irradiated DNA molecules in addition to their well known scavenging capacity. MATERIALS AND METHODS: Native and fragmented calf thymus DNA molecules were exposed to various doses of 60Co gamma-rays at approximately 4.5Gy/min. Using thermal transition spectrophotometry, pulsed field gel electrophoresis and standard agarose gel electrophoresis, the effects of Tris, phenol and NaCl on the double helix to single coil thermal transition temperature, Tm, and the yield of the double-strand breaks (Gdsb) of the irradiated DNA molecules have been studied. RESULTS: DNA molecules exposed to gamma-rays showed a decreased Tm and a corresponding increase of the Gdsb yield. Tris, as well as phenol, exhibited a strong protection against preventing these radiation-induced alterations. In addition, both substances strongly affected the thermal stability of the non-irradiated DNA samples. These results, compared with data obtained by NaCl and its effects on DNA thermostability and Gdsb, revealed that in the presence of both scavengers the observed dsb decrease was correlated to an increased molecular stability of DNA. CONCLUSIONS: This work suggests that the total protective effect of Tris and phenol against radiation-induced dsb is mainly attributed to their well-known radical scavenging properties, while relatively minor protective effects arise from their contribution to an increased molecular stability of DNA.

Alpha-particle-induced changes in the stability and size of DNA.

The effect of alpha-particle radiation on the thermal stability and size of calf thymus DNA molecules in deoxygenated aqueous solutions was investigated by thermal transition spectrophotometry, pulsed-field gel electrophoresis, and standard agarose gel electrophoresis. The thermal transition of DNA from helix to coil was studied through analysis of the UV A(260) absorbance. The results obtained for alpha particles of mean LET of 128 keV microm(-1) reveal a dual dose response: a tendency for thermal stability of the DNA helix at "low" doses, followed by an increasing instability at higher doses. The same phenomenon was observed for the mean molecular weight of DNA molecules exposed to alpha particles. The results reported here for alpha particles in the low-dose region of 0-16 Gy are consistent with our previous hypothesis of inter- and intramolecular interactions of a covalent character in gamma-irradiated DNA molecules in the dose region of 0-4 Gy.