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The fluorometric analysis of DNA unwinding (FADU assay) was originally designed for rapid detection of X-ray-induced DNA damage in mammalian cells. This cellular bioassay is based on time-dependent alkaline denaturation of DNA under moderate denaturing conditions (pH 12.2-12.4) starting from ends as well as from all DNA break points (single-strand breaks, SSB; double-strand breaks, DSB; alkali-labile sites, ALS). DNA which remained double-stranded after 30 min of alkaline treatment was detected after neutralisation and immediate fragmentation followed by binding to the Hoechst 33258 dye (bisbenzimide) and fluorescence measures. In the current paper, a modified method was used which allows cell cultivation and chemical treatment in the same microplate (micro-FADU) followed by analysis of 96 samples in a microplate fluorescence reader. Exposure of mammalian cells to chemicals was performed for 60 min on ice thus allowing identification of direct acting substances capable of inducing DNA-strand breaks. As an inter-assay standard the action of hydrogen peroxide was tested in every test plate. The results demonstrate that the micro-FADU assay is suitable to detect the presence of chemically induced strand breaks within 3 h.

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).

Calibration of the non-denaturing (pH 9.6) filter elution technique by 125I decays has been used to examine the relationship between X-ray-induced DNA double-strand breaks (dsb) and cellular survival of CHO cells and human fibroblasts. DNA dsb induction varies markedly between the two cell lines, reflecting the different survival levels. The relationships between lethal lesions (-ln S) and DNA dsb are also significantly different for CHO cells and human fibroblasts, suggesting either different probabilities of dsb conversion into lethal lesions or different repair capacities.

The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, was studied on DNA synthesis, PLD-recovery and DNA double-strand break rejoining in X-irradiated human fibroblasts. In unirradiated, exponentially growing cells, aphidicolin (0.5-5 micrograms ml) inhibited DNA synthesis almost completely. This effect depended not only on aphidicolin concentration but also on the duration of pre-incubation. The action of aphidicolin was found to be reversible. When aphidicolin had been removed, colony forming ability was not affected in aphidicolin pretreated cells. Aphidicolin pretreated and irradiated cells showed a reduction in PLD-recovery, dependent on aphidicolin concentration and duration of pretreatment. The initial number of DNA double-strand breaks (calibrated by 125I decay) was not affected by aphidicolin. However, after incubation for 90 min in the presence of aphidicolin there was a large reduction in double-strand break rejoining. With long incubation periods in aphidicolin rejoining was almost completely inhibited.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ−1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV μ−1 no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification. [Copyright &y& Elsevier]