A NOVEL TECHNIQUE FOR THE DETECTION OF DNA SINGLE-STRAND BREAKS IN HUMAN WHITE BLOOD-CELLS AND ITS COMBINATION WITH THE UNSCHEDULED DNA-SYNTHESIS ASSAY

A modified assay for the detection of DNA single-strand breaks (SSBs) in human mononucleated white blood cells (MWBCs) based on the nick translation (NT) reaction was developed and combined with the test for unscheduled DNA synthesis (UDS). Both assays were performed on disposable 96-well filtration plates and therefore allowed rapid and sensitive examination of SSBs and UDS. Only 5-8 ml of heparinized blood is required for an eightfold determination in both assays. The uptake of radioactive nucleotide precursors was demonstrated to depend linearly upon the NT reaction time and in both assay systems on the number of investigated cells. The best results and the lowest signal to noise ratio were obtained when the NT assay was performed at 25-degrees-C for 20 min. The test was standardized for 150000 MWBCs/well and a polymerase I concentration of 20 U/ml. The same number of cells were used to measure UDS during a 4-h incubation at 37-degrees-C. We observed a dose-dependent increase in SSBs after in vitro incubation with N-methyl-N-nitrosoguanidine (MNNG), with a detection limit of 50 muM when MNNG was present for 1 h and of 5 muM after 20-h incubation period. UDS in MWBCs was increased after treatment for 1 h with MNNG (200 muM) only if poly(ADP)ribose synthesis was inhibited by 3-aminobenzamide. UDS was induced by 320 muM methyl methanesulfonate, but SSBs could only be detected after inhibition of UDS by 100 muM hydroxyurea. The described modification of the NT procedure for the detection of SSBs in DNA of human MWBCs and its combination with the detection of UDS could serve as a useful tool for biological monitoring in occupational or environmental medicine.