|
D. A. Kurinnyi1, O. V. Zemskova2, M. G. Romanenko1, L. V. Neumerzhitska1, M. Yu. Makarchuk3,
O. E. Kutsyk4, S. R. Rushkovsky3
1 State Institution «National Research Center for Radiation Medicine, Hematology and Oncology of the
National Academy of Medical Sciences of Ukraine», 53 Yuriia Illienka Str., Kyiv, 04050, Ukraine
2 State Institution «Romodanov Institute of Neurosurgery of the National Academy of Medical Sciences of
Ukraine», 32 Platona Maiborody Str., Kyiv, 04050, Ukraine
3 Educational and Scientific Center «Institute of Biology and Medicine» of the Taras Shevchenko Kyiv
National University, 64/13 Volodymyrska Str., Kyiv, 01601, Ukraine
4 National Specialized Pediatric Hospital «OKHMATDYT» of the Ministry of Health of Ukraine, 28/1
Vyacheslava Chornovola Str., Kyiv, 01135, Ukraine
ANALYSIS OF DNA METHYLATION CHANGES IN MANIFESTATION OF DIRECT AND RESCUE BYSTANDER EFFECTS
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.
Problems of Radiation Medicine and Radiobiology.
2024;29:199-212. doi: 10.33145/2304-8336-2024-29-199-212
 full text
|
