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Human bone marrow mononuclear cells (BMMNCs) were separated by density gradient centrifugation, and a subpopulation of progenitor cells was further isolated using anti-CD34-coated magnetic beads. The cells were irradiated with gamma-rays (0.93-5.43 Gy) from a 137Cs source. The extent of DNA damage, i.e., single-strand breaks (SSBs) and alkali-labile lesions of individual cells, was investigated using the alkaline single-cell gel electrophoresis technique. The irradiation resulted in a dose-dependent increase in DNA migration, reflecting the number of detectable DNA lesions. An approximately similar extent of SSB formation was observed in BMMNCs and CD34 + cells. Damage was repaired when the cells were incubated at 37 degrees C: a fast initial repair phase was followed by a slower rejoining of SSBs in both BMMNC and CD34 + cell populations. A significantly longer time was required to repair the lesions caused by 5.43 Gy than those caused by 0.93 Gy. In the present work we report, for the first time, the induction and repair of DNA SSBs at the level of single human bone marrow cells when exposed to ionizing radiation at clinically relevant doses. These data, together with our previous results with human blood granulocytes and lymphocytes, indicate an approximately similar extent of formation and repair of gamma-irradiation-induced DNA SSBs in immature and mature human hematopoietic cells.

Human bone marrow mononuclear cells were isolated by density gradient centrifugation and irradiated with a 137Cs source. The extent of irradiation-induced single-strand breaks (SSBs) and alkali labile sites as well as their repair was investigated by using the alkaline single-cell gel electrophoresis (SCGE) technique, or comet assay. A dose-dependent increase in the length of DNA migration was seen when cells were exposed to 0, 2.43 and 5.43 Gy of gamma-irradiation. Complete repair of DNA SSBs was observed over 24 h after a dose of 2.43 Gy. Second challenges of 0, 2.43 and 5.43 Gy resulted in similar SSBs as with the first irradiation. Furthermore, the DNA repair kinetics of two cell populations, one previously unirradiated and the other having received 2.43 Gy 24 h earlier, was indistinguishable. This means that most human bone marrow cells retain their genetic stability after a dose of 2.43 Gy if SSBs are used as an endpoint.

Ionizing irradiation and UV-irradiation cause DNA damage. Ionizing irradiation induces single-strand breaks, much less abundantly double-strand breaks, alkali-labile sites, and various oxidized purines and pyrimidines. UV-irradiation, on the other hand, causes cyclobutane pyrimidine dimers, (6-4) photoproducts, and various monomeric base damages. The deposition of energy in DNA may result directly in single-strand breaks (predominant form after ionizing radiation), or the strand breaks may be generated during the repair process (predominant form after UV-irradiation). We investigated the formation and repair of DNA single-strand breaks in human blood granulocytes and lymphocytes by the single-cell gel electrophoresis or comet assay. The induction and repair of DNA lesions by gamma-irradiation was comparable in human blood granulocytes and lymphocytes. The finding is consistent with the expression of the pertinent base excision repair proteins in these cells. In contrast to gamma-irradiation, fewer single-strand breaks were observed immediately after UV-irradiation; the maximum number of breaks were seen when the cells were incubated for 30-60 min. After an incubation period of 150 min, a significant reduction of single-strand breaks was noted. It is conceivable that the first 30-60 min represented a period during which the incision-excision phase of nucleotide excision repair (NER) predominated. After that, strand joining was dominant, evidently representing the synthesis and ligation phase of NER. These results indicate that the approx. 30 different polypeptides required for complete NER are functional in these mature blood cells. This is the first demonstration of the expression of global NER in human granulocytes.