Three-Color FISH Method: Dose-Effect Curves for Translocations in Peripheral Blood Lymphocyte Cultures after Gamma-Irradiation In Vitro

Abstract

Purpose: Plotting dose-effect curves for translocations identified using the tricolor FISH method based on the results of cytogenetic analysis of cultures of peripheral blood lymphocytes of healthy donors after in vitro gamma irradiation. Material and methods: Venous blood was obtained from three donors (2 men and 1 woman aged from 28 to 41 years) and subjected to in vitro gamma irradiation from a 60Co source at doses of 0.10; 0.15; 0.25; 0.35; 0.50; 0.75; 1.00; 1.50; 2.00 and 3.00 Gy at 37 ° C (dose rate 0.5 Gy / min). For tricolor FISH staining, two different sets of DNA probes were used for chromosome pairs 1, 4, 12 and 2, 3, 8. Metaphases with a quasi-diploid number of chromosomes (40-46) and a complete set of all FISH-stained chromosomes, taking into account their total length, were selected for analysis. Differentiation of stable and unstable cells was also carried out. In the cytogenetic analysis, traditional terminology was used with the designation of translocations as reciprocal (complete, two-sided), non-reciprocal (terminal, incomplete, or unilateral), or interstitial. Results: The obtained numerical data were used to statistically compare the frequencies of FISH-recorded translocations when using different sets of DNA probes, when calculating of chromosome aberrations were in all (unstable and stable) and stable metaphase cells, when comparing of the frequencies of FISH-recorded translocations and dicentrics, and assessing of the contribution of the level of translocations between FISH-stained chromosome pairs in the total translocation frequency. The plotted dose-effect curves generally corresponded to the linear-quadratic form. Conclusion: Dose dependences obtained for translocations using two different selected tricolor sets of DNA probes did not differ statistically significantly. At the same time, cytogenetic analysis of only stable metaphase cells revealed a tendency to register lower levels of translocations than when analyzing all cells (unstable and stable ), at the highest doses of 2 and 3 Gy. The levels of dicentrics formed with the participation of FISH-stained chromosomes were significantly lower than the number of observed translocations. The quantitative contribution of translocations between FISH-stained pairs of chromosomes turned out to be very low, which clearly does not contribute to an increase in the sensitivity of the FISH method of retrospective dose estimation as compared to its one-color version. At the same time, the three-color FISH-staining makes it possible to identify such variants of chromosomal rearrangements that are not recorded using the one-color FISH method.