VALIDATION OF CHROMOSOME PAINTING AS A BIODOSIMETER IN HUMAN PERIPHERAL LYMPHOCYTES FOLLOWING ACUTE EXPOSURE TO IONIZING-RADIATION IN-VITRO

Fluorescence in situ hybridization with chromosome-specific composite DNA probes ('chromosome painting') appears to be a useful tool for quantifying symmetrical cytogenetic damage. However, a thorough comparison between chromosome painting and the conventional methods of GTG-banding and dicentric analysis has not been performed. We have undertaken the validation of chromosome painting using human blood exposed in vitro to Cs-137 gamma-rays at doses ranging from 0 to 400 cGy, then cultured according to standard procedures and harvested at 52h. For painting, bound probes were detected either with fluoresceinated avidin and counterstained with propidium iodide, or with ChromoBlue WCP Probe and Giemsa. The first approach utilizes ultraviolet excitation in which painted chromosomes appear yellow and the remaining chromosomes appear red. The ChromoBlue labelling approach requires ordinary light microscopy in which painted chromosomes appear dark blue and the remaining chromosomes appear light blue. With each method, exchanges between painted and unpainted chromosomes appear bicoloured. Because only a fraction of all possible exchanges are detected, the number of metaphases examined is adjusted according to the fraction of the genome painted. We have performed painting by several methods, including fluorescence with chromosome 4 probe alone, fluorescence with probes for chromosomes 1, 3 and 4 simultaneously, and chromogenic painting with chromosome 4 probe alone. The results obtained by the various painting methods were compared with GTG-banded cells which were examined for both translocations and dicentrics. In addition, unbanded metaphases stained with Giemsa were scored for dicentrics. Our data show that the frequency of chromosome exchanges detected by painting and banding agree with each other and with the number of dicentrics seen in unbanded cells, at least at doses of less-than-or-equal-to 200 cGy.