Andrey Osipov

Although ionizing radiation quality shapes the spatial structure and complexity of primary DNA lesions, its contribution relative to absorbed dose in the late DNA damage response (DDR) remains poorly quantified. We analyzed γH2AX and pATM foci kinetics in human mesenchymal stromal/stem cells exposed to ⁶⁰Co γ-rays, 150 MeV protons, and 14.1 MeV neutrons at doses of 0.25, 0.5, 1.0, and 2.0 Gy, delivered at dose rates of 0.5 Gy/min for γ-rays, 1 Gy/min for protons, and 46 mGy/min for neutrons, respectively. An effective kinetic model was used to describe the time course of the DDR and incorporated an ultraslow component as a model-based index of long-lasting repair burden. Radiation types differed less in early signal amplitude than in decay rate and late persistence. At every investigated dose, the normalized ultraslow fraction followed the order γ-rays < protons < neutrons. This ordering was preserved in per-dose fits, while parametric bootstrapping supported the γ–neutron separation, although the absolute values of the internal fractions were only partially identifiable. RAD51, analyzed independently from model fitting, showed the same qualitative ordering at late time points, supporting the interpretation that radiation quality is more associated with persistent DDR signaling than absorbed dose within the investigated range. These findings suggest that absorbed dose alone may not fully characterize the late cellular response to different radiation qualities and highlight the importance of radiation quality in comparative radiobiology, radiation protection, and the interpretation of normal-tissue responses to therapeutic irradiation.

Laser-driven accelerated particle beams have significantly advanced cancer treatment by facilitating the delivery of exceptionally high dose rates of radiation to solid tumors within femto- to picosecond timescales. This investigation compared the radiobiological effectiveness of ultrashort pulsed electron beams, generated by the Advanced Research Electron Accelerator Laboratory (AREAL) accelerator, with conventional X-rays on two non-small cell lung cancer (NSCLC) cell lines: A549 (wild-type p53) and H1299 (p53-deficient). NSCLC cells were irradiated using either the AREAL accelerator (with a peak dose rate of 1.6 × 10¹⁰ Gy/s, a pulse duration of 4.5 × 10⁻¹³ s, and a repetition rate of 20 Hz) or an X-ray unit at an absorbed dose rate of 0.85 Gy/min. Clonogenic survival analysis, γH2AX foci enumeration, and genome-wide transcriptome analysis were conducted. Clonogenic survival curves showed increased radiosensitivity of A549 cells following AREAL exposure compared to X-rays (RBE = 1.2), whereas H1299 radiosensitivity remained unchanged. In both cell lines, AREAL exposure resulted in a greater dose-dependent accumulation of residual γH2AX foci 24 h after irradiation than conventional X-rays, suggesting more persistent DNA damage signaling. Transcriptomic analyses revealed broader gene expression changes after AREAL irradiation and suggested distinct p53-related responses. Pathway-level analysis demonstrated that A549 cells exhibited reduced DNA repair activity, accompanied by dysregulation of cell cycle progression and apoptosis, whereas H1299 cells displayed transcriptomic signatures consistent with enhanced homologous recombination activity. Overall, these findings indicate that ultrashort pulsed electron beams induce p53-related responses distinct from those of conventional X-rays and warrant further investigation of this technology as a potential radiotherapy modality.

Laser-driven accelerated particle beams have revolutionized the field of cancer treatment by enabling the delivery of extremely high dose-rates of radiation to solid tumors in a matter of femto- to picoseconds. In this study, radiobiological effectiveness of ultrashort pulsed electron beams generated by Advanced Research Electron Accelerator Laboratory (AREAL) accelerator and conventional X-rays were compared on two non-small cell lung cancer (NSCLC) cell lines, A549 (wild-type p53) and H1299 (p53-deficient). NSCLC cells were irradiated at using a AREAL accelerator (a peak dose rate of 1.6 × 1010 Gy/s, pulse duration of 4.5 × 10–13 s, repetition rate of 20 Hz) or X-ray unit at an absorbed dose rate of 0.85 Gy/min. Clonogenic survival analysis, γH2AX foci enumeration and genome-wide transcriptome analysis were performed. Clonogenic survival curves showed increased ra-diosensitivity of A549 cells after AREAL exposure compared to X-rays (RBEs=1.2), while H1299 radiosensitivity has not changed. AREAL exposure caused higher linear dose–dependent increase in the number of residual γH2AX foci in both A549 and H1299 cells compared to X-rays. The DNA double-strand breaks in H1299 cells were mainly repaired through homologous recombination following AREAL exposure. However, the gene ex-pression analysis of pathway activation levels revealed a down-regulation of the same pathways in A549 cells. This resulted in activation of Integrin-linked kinase-dependent apoptosis, G2 and proliferation arrest of A549 cells.

The growing use of computed tomography (CT) in medicine requires a better understanding of how low-dose radiation affects human stem cells. This study investigated the long-term consequences of CT-level radiation on the secretory profile of human adipose-derived mesenchymal stem cells (AD-MSCs). AD-MSCs were exposed to radiation regimens simulating a single or multiple head CT scans, or to a single 2 Gy therapeutic dose, and their secretion of 41 cytokines, chemokines, and growth factors was monitored during long-term culture. At the early passage, AD-MSCs receiving a single 2 Gy dose showed a coordinated increase in several lymphocyte-regulating cytokines compared to cells exposed to multiple CT scans. However, these initial differences were not sustained. Long-term culturing led to a progressive and widespread decrease in the secretion of 26 cytokines, chemokines, and growth factors across all groups. By the latest passage, all irradiated cells showed a generalized reduction in secretory function compared to non-irradiated controls. These findings demonstrate that while different radiation regimens trigger distinct immediate responses, long-term culture results in a broad decline of the AD-MSCs secretome, which is accentuated by prior radiation exposure. This underscores the importance of assessing long-term consequences to fully evaluate the functional impact of diagnostic radiation on stem cells.

An open question in radiobiology concerns whether low doses of radiation are harmful or if cells are able to tolerate such exposure with minimal or no disruption. This issue is relevant for evaluating public health risks associated with the increasing number of medical computed tomography (CT) diagnostic procedures. This study evaluated the impact of CT scan-level exposure on human adipose mesenchymal stem cells (hMSCs) by measuring DNA damage responses (γH2AX, 53BP1, pATM foci), proliferation (Ki-67), senescence (β-galactosidase), and multiple gene expressions. Responses to one or five CT exposures were compared to a 2 Gy X-ray dose at intervals from 1 h to 10 passages post-irradiation. It was shown that CT scan briefly increased DNA damage markers but showed no significant long-term effects. A high dose of 2 Gy X-ray exposure caused sustained DNA damage, decreased proliferation, increased senescence, and significant changes in hundreds of genes even after several cell generations. After a single CT exposure, gene expression changes were minimal, while high-dose exposure led to strong activation of DNA repair and stress response pathways. Five CT scans caused a slight activation of LIF and HSPA1B genes, but these effects were minor compared to the high-dose group. All detected effects from CT scans were not observed by ten cell passages, whereas high-dose effects persisted. In conclusion, typical CT scan exposures have only short-term, mild effects on hMSCs, while high-dose radiation causes lasting cellular and genetic changes.

The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.

Understanding the relative contributions of different repair pathways to radiation-induced DNA damage responses remains a challenging issue in terms of studying the radiation injury endpoints. The comparative manifestation of homologous recombination (HR) after irradiation with different doses greatly determines the overall effectiveness of recovery in a dividing cell after irradiation, since HR is an error-free mechanism intended to perform the repair of DNA double-strand breaks (DSB) during S/G2 phases of the cell cycle. In this article, we present experimentally observed evidence of dose-dependent shifts in the relative contributions of HR in human fibroblasts after X-ray exposure at doses in the range 20–1000 mGy, which is also supported by quantitative modeling of DNA DSB repair. Our findings indicate that the increase in the radiation dose leads to a dose-dependent decrease in the relative contribution of HR in the entire repair process.

Relative contribution of different repair pathways to the radiation-induced DNA damage responses remains a challenging issue of studying the radiation injury endpoints. Comparative manifestation of homologous recombination (HR) after application of different radiation doses greatly determines an overall effectiveness of recovery in dividing cell after irradiation, since HR is an error-free mechanism intended for repair of DNA double strand breaks (DSB) during S/G2 phases of cell cycle. In this article, we present an experimentally observed evidence of dose-dependent shift in relative contribution of HR in human fibroblasts after X-ray exposure at doses 20-1000 mGy, which is also supported by quantitative modeling of DNA DSB repair. Our findings indicate that the radiation dose increase leads to dose-dependent decrease in relative contribution of HR into the entire repair process.

DNA repair (DNA damage) foci observed 24 h and later after irradiation are called “residual” in the literature. They are believed to be the repair sites for complex, potentially lethal DNA double strand breaks. However, the features of their post-radiation dose-dependent quantitative changes and their role in the processes of cell death and senescence are still insufficiently studied. For the first time in one work, a simultaneous study of the association of changes in the number of residual foci of key DNA damage response (DDR) proteins (γH2AX, pATM, 53BP1, p-p53), the proportion of caspase-3 positive, LC-3 II autophagic and SA-β-gal senescent cells was carried out 24–72 h after fibroblast irradiation with X-rays at doses of 1–10 Gy. It was shown that with an increase in time after irradiation from 24 h to 72 h, the number of residual foci and the proportion of caspase-3 positive cells decrease, while the proportion of senescent cells, on the contrary, increases. The highest number of autophagic cells was noted 48 h after irradiation. In general, the results obtained provide important information for understanding the dynamics of the development of a dose-dependent cellular response in populations of irradiated fibroblasts.