Exposure to ionizing and non-ionizing radiation from environmental and clinical settings can significantly threaten genomic stability, especially when combined. This ex vivo study investigates the potential combined effects of gamma radiation and ultraviolet B (UVB) exposure on human peripheral blood mononuclear cells (PBMCs) from healthy donors by exposing whole blood and isolated PBMCs to 1 Gy of gamma rays, to an absolute dose of approximately 100 J/m² of UVB, or to their combination. Combined exposure resulted in significantly elevated γH2AX foci formation and chromosomal aberrations relative to individual stressors, with the most pronounced effects observed in isolated PBMCs. Notably, lymphocytes from some donors failed to proliferate after UVB or co-exposure. Based on our results, a predictive biophysical model derived from dicentric yield was developed to estimate the gamma-ray equivalent dose from co-exposure, indicating up to ~9% increase in lifetime cancer risk. Although this proof-of-concept study included only a small number of donors and focused on two endpoints (γH2AX and dicentric assays), it provides a controlled framework for investigating mechanisms of radiation-induced genomic instability. The results emphasize the importance of accounting for mixed radiation exposures in genotoxic risk assessment and radiation protection.

Proton therapy offers superior dose localization, yet the biological effects of low-energy protons relevant to superficial tissues remain underexplored. We report the design and validation of a proton irradiation setup developed at the Tandem Accelerator of NCSR “Demokritos” for controlled radiobiological experiments. Monte Carlo simulations using Geant4 and Monte Carlo Damage Simulation (MCDS—Monte Carlo Damage Simulation) were used to determine proton energy spectra, linear energy transfer (LET), and predicted DNA damage yields. A single layer (15–20 μm in thickness) of human keratinocytes (HaCaT) was irradiated at doses from 0.65 to 3.65 Gy, and γ-H2AX foci were quantified as markers of tracks including one or more DNA double-strand breaks. The system achieved a uniform dose rate of 0.37 Gy/min, as calculated with Geant4, with a mean proton energy of 4.1 MeV (LET ≈ 8 keV/μm). A strong correlation (R² = 0.93) was observed between proton dose and γH2AX foci per nucleus (~10 foci/Gy), reflecting damage-inducing proton tracks rather than individual DNA double-strand breaks. At higher doses, an increased fraction of cells exhibited pan-nuclear γH2AX staining, characterized by a diffuse γH2AX signal throughout the nucleus and commonly associated with extensive or clustered DNA damage and global chromatin phosphorylation. These responses are consistent with the well-established dense ionization patterns produced by low-energy protons, as indicated by the LET spectrum and supported by MCDS-predicted clustered damage yields. While the γH2AX assay does not directly resolve simple versus complex DNA lesions, the agreement between Monte Carlo modeling and the observed cellular stress responses indicates that the irradiation platform reliably reproduces the expected biological signatures of low-energy proton exposure. Consequently, the developed system provides a robust experimental tool for systematic investigations of cellular radiosensitivity and radiotoxicity, with potential applications in skin dosimetry and radioprotection.

Exposure to ionizing and non-ionizing radiation from environmental and clinical settings can significantly threaten genomic stability, especially when combined. This ex vivo study investigates the potential combined effects of gamma radiation and ultraviolet B (UVB) light on human peripheral blood mononuclear cells (PBMCs) from healthy donors by exposing whole blood and isolated PBMCs to 1 Gy of gamma rays, 100 J/m² of UVB, or to their combination. DNA damage and repair were assessed using the γH2AX immunofluorescence assay at 1 hour and 24 hours post-irradiation. Combined exposure resulted in significantly elevated γH2AX foci formation and chromosomal aberrations relative to individual stressors, with the most pronounced effects observed in isolated PBMCs. Notably, lymphocytes from some donors failed to proliferate after UVB or co-exposure. Based on our results, a predictive biophysical model derived from dicentric yield was developed to estimate the gamma-ray equivalent dose from co-exposure, indicating a potential ~9% increase in lifetime cancer risk. These findings highlight the need to account for mixed radiation exposures in genotoxic risk assessment and radiation protection, while also supporting a protective role of the whole-blood environment.