Peripheral blood lymphocytes differ in DNA damage response after exposure to X-rays with different physical properties

AbstractIntroduction: 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: LymphocytesDNA damageDNA repairmicronucleus assayɣ-H2AX focicell deathX-ray beam qualitydose rate AcknowledgementsWe would like to thank L. Pieters, G. De Smet, J. Aernoudt, E. Bes, T. Thiron for technical assistance. Furthermore, we acknowledge E. Duthoo and E. Beyls for their help with MN scoring and I. Efimova, for assistance with cell death measurements. We would like to thank Klaus Bacher and his Medical Physics team, in particular P.J. Kellens and T. Gossye, for their aid in the dosimetry of the SARRP. Also, we thank P.J. Kellens for aid during statistical analysis of our data. Thanks to the group of Luc Van Hoorebeke for the discussion meeting and providing the copper layers.Author contributionsS.S., A.V., D.K. and A.B.: conceptualization and methodology of the experiments. S.S., L.D., F.V. and R.D. performed the lab experiments. S.S., A.B., D.K. and B.V. wrote the manuscript, revised and prepared the final version. S.S., A.B., B.V., C.D., K.B. performed dosimetry and prepared beam set-ups. A.B. and D.K. supervised and were responsible for funding acquisition. All authors have read and agreed with the published version of the manuscript.Disclosure statementNo potential conflict of interest was reported by the author(s).Additional informationFundingThis work was supported by the [Bijzonder onderzoeksfonds Ghent University] under Grant (BOFSTA20170018; BOF/CDV/2022/0007)' to A.B. and by the [Fonds voor wetenschappelijk onderzoek] under Grant [FWO, G051918N] funding to A.B., A.V and D.K. The "Cell Death Investigation and Therapy" (CDIT) laboratory was supported by FWO-Flanders Research Grants under Grant [G043219N and G016221N], and by the [Ghent University Grants] under [BOF01/O3618; BOF/IOP/2022/033; BOF23/GOA/029] and has received funding from the [FWO and F.R.S.-FNRS under the "Excellence of Science" (EOS) program] under Grant [G0I5322N]. Robin Demuynck is a Ph.D. student in the CDIT laboratory and holds a Ph.D. fellowship from [FWO-Flanders] under Grant [11E3121N]. Notes on contributorsSimon SioenSimon Sioen obtained a Master of Science in Biomedical Sciences in 2018 at Ghent University, Belgium. He started his PhD in September 2018 at the Radiobiology research group at Ghent University.Louise D'HondtLouise D'Hondt obtained her Master of Science in Biomedical Sciences, major Medical Radiation Sciences, summa cum laude at the University of Ghent, Belgium in 2022. In November 2022, Louise started a joint PhD under the supervision of prof. Klaus Bacher (PhD, Ir) and prof. Annemiek Snoeckx (PhD, MD) at respectively Ghent University and the University of Antwerp.Fien Van HouteFien Van Houte obtained a Master of Science in Biomedical Sciences cum laude in 2021 at Ghent University. During this period, Fien performed research at the radiobiology department for her master's dissertation: 'DNA damage response after exposure to different X-ray beam qualities.Robin DemuynckRobin Demuynck obtained his Master of Science in Biomedical Sciences magna cum laude (in 2020) at the University of Ghent, Belgium.He started his PhD under the supervision of prof. Dmitri Krysko (MD, PhD). In September 2020 he received the FWO PhD Fellowship for Fundamental Research.Klaus BacherKlaus Bacher is a medical physics expert. He is professor and head of the division of Medical Physics at the Faculty of Medicine and Health Sciences of the Ghent University. His research interests mainly focus on dose-image quality assessments of new diagnostic imaging modalities. He is the past-president of the European Alliance for Medical Radiation Protection Research (EURAMED).Carlos De WagterCarlos De Wagter was a senior full professor in the domain of medial radiation physics (2012-2021) at Ghent University. He was also Chairman of University Department Human Structure and Repair (2018-2021) and he is now a professor emeritus.Anne VralAnne Vral is full professor and principal investigator of the Radiobiology Research Group, Ghent University, Belgium. She has 30 years of experience in the field of basic and radiobiology, biological dosimetry and cancer. The topics related to cancer are dealing with radiosensitivity and DNA repair.Barbara VanderstraetenBarbara Vanderstraeten is M.Sc. in Engineering Physics and obtained a Ph.D. in Medical Sciences. She is an assistant professor at the Department of Human Structure and Repair at the Faculty of Medicine and Health Sciences in Ghent University and medical physics expert at the Department of Radiotherapy-Oncology at Ghent University Hospital. Her research interests are focused on radiotherapy treatment planning and dose calculation, automated planning, biological imaging, quality assurance and the economic evaluation of new radiotherapy techniques.Dmitri V. KryskoDmitri V. Krysko is an Associate Professor and the head of the Cell Death Investigation and Therapy (CDIT) Laboratory at the Department of Human Structure and Repair, Faculty of Medicine and Health Sciences at Ghent University (UGent). He obtained his Ph.D. in 2006 at UGent (Belgium) and an M.D. in 1998 from Saratov State Medical University (Russia). Prof. Krysko aims to understand the role of immunogenic cell death in cancer and develop novel experimental cancer immunotherapy. For research on immunogenic cell death, he received the GSK Vaccine 2019 Award given by the Royal Belgian Academy of Medicine and AstraZeneca Foundation 2019 Award.Ans BaeyensAns Baeyens was from 2005 until 2015 a postdoctoral researcher at iThemba LABS, South Africa and head of the Radiobiology lab at the University of Witwatersrand in Johannesburg, South Africa. Prof Baeyens is Associate professor at the Department of Human Structure and Repair at the faculty of Medicine and Health Sciences, Ghent University. She is principal investigator at the Radiobiology group and head of the reference lab for radiosensitivity testing in Belgium. Her research deals with chromosomal radiosensitivity, biological dosimetry and measuring biological effects after exposure to different types of ionizing radiation.