An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody (D1B) was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the time-dependent partial unwinding of cellular DNA under alkaline conditions. Single- and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding from these points under defined conditions is a measure of the number of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single-from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gy of ionizing radiation and needs only small numbers of cells. The usefulness of the technique was demonstrated in a study of the induction of DNA damage and its repair in cultured Chinese hamster cells and in human white blood cells after exposure to 60Co-gamma-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related DNA unwinding was observed and repair of DNA lesions was observed up to 60 min after irradiation.
The alkaline elution technique for the detection of DNA damage has been adapted to allow application on unlabelled blood cells. Both the induction and subsequent repair have been studied of two classes of DNA damage, viz, single-strand breaks and base damage recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of base damage after in vitro exposure of full blood under aerobic conditions to biologically relevant doses of gamma-rays (1.5-4.5 Gy). After a radiation dose of 3 Gy about 50% of the base damage was removed within 1.5 h of repair. Base damage could still be detected at 24 h after exposure to 15 Gy.
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