ANALYSIS OF DNA METHYLATION CHANGES IN MANIFESTATION OF DIRECT AND RESCUE BYSTANDER EFFECTS.

Abstract

Objective: to investigate changes in DNA methylation in bystander and inducer cells during the manifestation ofdirect and rescue bystander effects.

Methods: Separate and co-cultivation of peripheral blood lymphocytes (PBL) of 10 conditionally healthy individuals; γ-quantum irradiation (IBL-237C emitter); modified comet electrophoresis method (Comet assay) under neutralconditions using the methylation-sensitive restriction enzyme HpaII; fluorescence microscopy with an automatedcomputer software system for analyzing the results; statistical methods.

Results: The level of DNA methylation in PBL was quantitatively assessed using DNA migration parameters inagarose gel: the length of the comet tail (in μm), the percentage of DNA in the tail part of the comet, and TailMoment (TM), which simultaneously takes into account both the amount of DNA in the tail part of the comet andthe length of the tail. In separate cultivation of PBL irradiated with γ-quanta (dose 1.0 Gy, power 2.34 Gy/min),a reliable decrease in the average values compared to the non-irradiated control was noted for the length of the«comet» tail ((57.03 ∓ 1.17) μm versus (66.64 ∓ 2.03) μm; p < 0.001) and Tail Moment (67.77 ∓ 1.22 versus85.06 ∓ 2.30; p < 0.001), which may indicate a decrease in the number of restriction sites of the methyl-sensitiverestriction enzyme HpaII and, as a consequence, an increase in the level of global DNA methylation. When thebystander effect is realized, the level of DNA damage in bystander cells increases, while there is a significantdecrease in the average values of the following parameters: the percentage of DNA in the tail part of the «comets»(p<0.001), the length of the tail part (p<0.001) and TailMoment (p<0.001) compared to both the non-irradiatedcontrol and irradiated PBLs in separate cultivation, and indicates an increase in the level of global DNA methylation.As in irradiated lymphocyte cultures cultured separately, in inducer cells a reliable decrease in the mean values oftail length (p < 0.01) and TailMoment (p < 0.001) was observed compared to the control, which may indicate adecrease in the number of restriction sites and an increase in the level of global DNA methylation as a result of irradiation. Between inducer cells and irradiated cells that were cultured separately, no difference was found in themean values of all the studied parameters: the percentage of DNA in the tail part of comets (p > 0.05), tail length(p > 0.05) and TailMoment (p 0.05), which may indicate the absence of changes in the level of DNA methylationwhen a non-irradiated culture is exposed to an irradiated one during co-cultivation.

Conclusion: The development of the direct bystander effect is accompanied by epigenetic changes, which are characterized by an increase in the level of DNA methylation in bystander cells. At the same time, in inducer cells,changes in the level of DNA methylation were not determined, which indicates the absence of manifestations of thereverse bystander effect at the epigenetic level.