Introduction: In radiology, low X-ray energies (<140 keV) are used to obtain an optimal image while in radiotherapy, higher X-ray energies (MeV) are used to eradicate tumor tissue. In radiation research, both these X-ray energies being used to extrapolate in vitro research to clinical practice. However, the energy deposition of X-rays depends on their energy spectrum, which might lead to changes in biological response. Therefore, this study compared the DNA damage response (DDR) in peripheral blood lymphocytes (PBLs) exposed to X-rays with varying beam quality, mean photon energy (MPE) and dose rate.Methods: The DDR was evaluated in peripheral blood lymphocytes (PBLs) by the ɣ-H2AX foci assay, the cytokinesis-block micronucleus assay and an SYTOX-based cell death assay, combined with specific cell death inhibitors. Cell cultures were irradiated with a 220 kV X-ray research cabinet (SARRP, X-Strahl) or a 6 MV X-ray linear accelerator (Elekta Synergy). Three main physical parameters were investigated: beam quality (V), MPE (eV) and dose rate (Gy/min). Additional copper (Cu) filtration caused variation in the MPE (78 keV, 94 keV, 118 keV) at SARRP; dose rates were varied by adjusting tube current for 220 kV X-rays (0.33-3 Gy/min) or water-phantom depth in the 6 MV set-up (3-6 Gy/min).Results: The induction of chromosomal damage and initial (30 min) DNA double-stranded breaks (DSBs) were significantly higher for 220 kV X-rays compared to 6 MV X-rays, while cell death induction was similar. Specific cell death inhibitors for apoptosis, necroptosis and ferroptosis were not capable of blocking cell death after irradiation using low or high-energy X-rays. Additional Cu filtration increased the MPE, which significantly decreased the amount of chromosomal damage and DSBs. Within the tested ranges no specific effects of dose rate variation were observed.Conclusion: The DDR in PBLs is influenced by the beam quality and MPE. This study reinforces the need for consideration and inclusion of all physical parameters in radiation-related studies.
Keywords: DNA damage; DNA repair; Lymphocytes; X-ray beam quality; cell death; dose rate; micronucleus assay; ɣ-H2AX foci.