ANALYSIS OF DNA METHYLATION CHANGES IN MANIFESTATION OF DIRECT AND RESCUE BYSTANDER EFFECTS
Authors:   State Institution «National Research Center for Radiation Medicine, Hematology and Oncology of the National Academy of Medical Sciences of Ukraine», Kurinnyi D. A. , Zemskova O. V. ,   State Institution «Romodanov Institute of Neurosurgery of the National Academy of Medical Sciences of Ukraine», Romanenko M. G. ,   State Institution «National Research Center for Radiation Medicine, Hematology and Oncology of the National Academy of Medical Sciences of Ukraine», Neumerzhitska L. V. ,   State Institution «National Research Center for Radiation Medicine, Hematology and Oncology of the National Academy of Medical Sciences of Ukraine», Makarchuk M. Yu. ,   Educational and Scientific Center «Institute of Biology and Medicine» of the Taras Shevchenko Kyiv National University, Kutsyk O. E. ,   National Specialized Pediatric Hospital «OKHMATDYT» of the Ministry of Health of Ukraine, Rushkovsky S. R. ,   Educational and Scientific Center «Institute of Biology and Medicine» of the Taras Shevchenko Kyiv National University
Published: December 2024

Objective: to investigate changes in DNA methylation in bystander and inducer cells during the manifestation of direct 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 neutral conditions using the methylation-sensitive restriction enzyme HpaII; fluorescence microscopy with an automated computer software system for analyzing the results; statistical methods.
Results. The level of DNA methylation in PBL was quantitatively assessed using DNA migration parameters in agarose gel: the length of the comet tail (in μm), the percentage of DNA in the tail part of the comet, and Tail Moment (TM), which simultaneously takes into account both the amount of DNA in the tail part of the comet and the 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 versus 85.06 ± 2.30; p < 0.001), which may indicate a decrease in the number of restriction sites of the methyl-sensitive restriction enzyme HpaII and, as a consequence, an increase in the level of global DNA methylation. When the bystander effect is realized, the level of DNA damage in bystander cells increases, while there is a significant decrease 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-irradiated control 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 of tail length (p < 0.01) and TailMoment (p < 0.001) was observed compared to the control, which may indicate a decrease 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 the mean 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 methylation when 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 the reverse bystander effect at the epigenetic level.
Key words: joint/separate cultivation of human lymphocytes; ionizing radiation; direct and reversed bystander effects; methyl-sensitive Comet assay.