Effect of gamma irradiation on DNA methylation profile in lymphocytes under whole blood culture conditions: An in vitro experimental study

Introduction. The expanding applications of ionizing radiation (IR) drive the improvement of methods to assess radiation exposure consequences. In this regard, deoxyribonucleic acid (DNA) is the most radiation-sensitive molecule: both DNA structure and epigenetic processes—specifically DNA methylation—are subject to change. Assessing DNA methylation levels helps register not only changes in gene methylation but also important cellular processes, such as DNA replication and repair and gene expression. IR exposure leads to an increase or decrease in gene methylation levels, as well as the activation of repair processes, apoptosis, and cell cycle regulation. DNA methylation levels also change as a result of cell culture conditions — gene expression increases or decreases depending on various factors: biological material (cell cultures, whole blood), cultivation time (3–24 h), irradiation frequency, and dose. Changes in DNA methylation levels after gamma irradiation followed by 48-hour cell culture remain insufficiently studied. Objective. To assess gene methylation levels before and after lymphocyte culture following a single gamma irradiation of whole blood in vitro . Materials and methods. The study was conducted using venous blood samples from nine apparently healthy men aged 24.6 ± 2.1 years. The blood samples were divided into aliquots designated for uncultured analysis or cell culture. Both types of aliquots were then allocated to either the 1.5 Gy group (exposed to in vitro gamma irradiation at a dose of 1.5 Gy) or the 0 Gy group (unirradiated control). The 0 Gy and 1.5 Gy groups within the culture subset were incubated for 48 h. Lymphocyte proliferation was stimulated by adding phytohemagglutinin (PHA). For the quantitative determination of CpG dinucleotide methylation levels, reduced representation bisulfite sequencing (RRBS) was applied using the XmaI restriction enzyme. Sequencing data were processed using the standard Ion Torrent Suite™ software and the rrbsData package. Calculations were performed using ROTS (reproducibility-optimized test statistic) and the R 3.6.3 programming language. Results. The levels of CpG dinucleotide methylation did not differ regardless of whether the lymphocytes were cultured, as seen in both the non-irradiated (0 Gy) and irradiated (1.5 Gy) groups. A comparison of IR effects on DNA methylation showed that in uncultured lymphocytes, CpG dinucleotide methylation showed no differences between the dose groups. In contrast, statistically significant changes in the methylation levels of 78 CpG dinucleotides (FDR, p < 0.05) were observed between the 0 Gy and 1.5 Gy groups following cell culture. Additionally, differences in Gene Ontology functional enrichment were found between hypo- and hypermethylated CpG dinucleotides following gamma irradiation. Conclusions. In the absence of lymphocyte cultivation, DNA methylation levels do not change after a single 1.5 Gy gamma irradiation of blood. The presence of cultivation (with PHA addition) for 48 h allows epigenetic changes to be detected following external in vitro gamma irradiation of blood.