Purpose: The objective of the study was to estimate the DNA damage in blood leukocytes at long terms after irradiation of mice with carbon ions (450 MeV/nucleon) both before and in the Bragg peak. Materials and methods: White outbred SHK male mice were exposed to whole-body irradiation with carbon ions at doses of 0.1–2 Gy in the spread-out Bragg peak and at a dose of 6 Gy before and in the Bragg peak. At different times after irradiation (1-75 days), whole blood was collected from the tail of each mouse and analyzed by the comet assay. Mice X-irradiated in the same dose range served as a positive control. The level of the expression of mRNA of CDKN1A, APEX1, BBC3, TXN2, and β-ACT genes in bone marrow cells was determined in animals irradiated with carbon ions at doses of 0.1–2 Gy using the real-time PCR. Results: It was found that, 24 h after ¹²C-irradiation, a dose-dependent (0.1–2 Gy) increase in the DNA damage of leukocytes occurred, which was accompanied by a decrease in their concentration and an increase in the expression of the CDKN1A and BBC3 genes in bone marrow cells. The expression of the APEX1 and TXN2 genes did not change. In mice ¹²C-irradiated at a dose of 6 Gy before and in the Bragg peak, the level of DNA damage changed as follows: by day 3, it increased; by day 23 it returned to the control level; by day 30, it increased again; and by day 75, it fell to the control level on irradiation before the Bragg peak and was significantly higher (р < 0.05) than in the control after irradiation in the Bragg peak. Conclusions: The dynamics of changes in the level of DNA damage in leucocytes of ¹²C-irradiated mice within 30 days is similar to that in mice exposed to sublethal doses of X-radiation. The retention of the high level of DNA damage by day 75 after ¹²C-irradiation in the Bragg peak indicates a significant injury of cells from different cell pools of the blood system. The high level of DNA damage may be related not only to complex DNA injuries but also to chronic oxidative stress.

In order to study the FLASH effect using live models, this work compared proton-induced damage to embryos (nine days after fertilization) and one-day-old chicks (18 days after fertilization) from irradiated at different dose rates eggs of Japanese quail (Coturnix coturnix japónica). Eggs were irradiated with protons in different modes depending on the dose rate: in a conventional mode (<1 Gy/s, CONV), in a flash mode (∼100 Gy/s, FLASH) and in a single-pulse flash mode (∼10⁵ Gy/s SPLASH). By the criteria of body weight and length, as well as the number of erythrocytes with micronuclei in nine-day-old embryos from eggs irradiated in the spread-out Bragg peak (SOBP) (8.5 Gy), FLASH and SPLASH modes were found to be less traumatic compared with the CONV mode. Among all irradiated embryos, the maximum body weight and length were observed in the SPLASH mode. The lowest death incidence and the smallest number of abnormal erythrocytes were recorded after FLASH and SPLASH irradiation. In chicks that hatched from eggs irradiated in the CONV mode, a tendency for an increase in the number of abnormal erythrocytes was observed. The speed of movement of chicks from FLASH- and SPLASH-irradiated eggs was comparable with that from unirradiated eggs, while chicks from eggs irradiated in the CONV mode were less active than all others. The proton irradiation of eggs in SOBP using high dose-rate modes is less damaging for healthy tissues and for the development of embryos and chicks on the cellular, anatomical, and physiological levels.

Among the methods for increasing the specificity of tumor radiotherapy, FLASH radiotherapy (FLASH-RT) stands out, having recently entered clinical trials. A distinctive feature of this treatment method is the delivery of a therapeutic dose in a fraction of a second with a typical mean dose rate greater than 40 Gy/s. In addition to improved patient comfort and a shorter hospital stay, this therapy potentially carries a lower risk of radiation-related side effects due to reduced damage to normal tissues. Numerous preclinical and in vivo laboratory trials of FLASH-RT have demonstrated that, in addition to reducing the severity of radiation-related complications, FLASH radiotherapy has antitumor efficacy similar to conventional radiotherapy. Partly reduced radiotoxicity after such a dose rate delivery obtained, in a broader radiobiological sense, an eponymous term FLASH effect. Although the first clinical trials aimed to evaluate the safety and efficiency of FLASH-RT against bone metastases (FAST-01/02), melanoma skin metastases (IMPulse, Flash-Skin I), Squamous Cell Carcinoma, or Basal Cell Carcinoma (LANCE) have already started or even finished and showed promising results (FAST-01), the radiobiological basis of the FLASH effect is far from a complete explanation. The fundamental factors explaining the nature of the FLASH effect are mainly considered to be the following: (1) changes in the balance of water radiolysis products and a decrease in the generation of stable reactive oxygen species (ROS), (2) differential oxygen depletion, depending on the initial oxygen concentration in tissues, and (3) physiological and metabolic, gene expression and probably epigenetic shifts in response to irradiation in normal and tumor cells. The main purpose of this review is the systematization of the radiobiological manifestations of the FLASH effect together with a consideration of the elementary processes laying in the basis of the FLASH effect in order to actualize rationale and future application developments of FLASH-RT.

The study of biological effects of high-dose-rate ionizing radiation using animal models remains an urgent problem. In the work, a comparison of proton-induced damage to embryos (the 9 ^th post-fertilization day) and 1-day-old baby chicks (the 18 ^th post-fertilization day) from irradiated eggs of Japanese quail ( Coturnix coturnix japónica ) irradiated in and outside the Bragg peak with the conventional and ultra-high dose rate D (CONV D < 1 Gy/s, FLASH D ∼100 Gy/s and single-pulse flash - SPLASH D ∼10 ⁶ Gy/s, respectively) was carried out. The following criteria were used: survival, body weight and body length of embryos, weight of chicks, percentage of erythrocytes with anomalies (with micronuclei, without nuclei, with two nuclei), and the speed of movement of 1-day-old chicks in the open field test. The largest number of dead embryos was recorded after irradiation with 7 and 14 Gy in the plateau region of the proton trajectory. By the criteria of body weight and length, as well as the number of erythrocytes with micronuclei in 9-day-old embryos from eggs irradiated with a dose of 8.5 Gy in the Bragg peak, FLASH and SPLASH modes were found to be the least traumatic compared with the CONV mode. The weight of chicks from eggs irradiated with a dose of 4 Gy in the Bragg peak did not differ from the control and did not depend on the irradiation mode. The lowest death incidence and the smallest number of abnormal erythrocytes were recorded after FLASH and SPLASH irradiation; in chicks that hatched from eggs irradiated in the CONV mode, a tendency for an increase in the number of abnormal erythrocytes was observed. The speed of movement of chicks from FLASH- and SPLASH-irradiated eggs was comparable with that from unirradiated eggs, and chicks from eggs irradiated in the CONV mode were less active than chicks from unirradiated eggs and eggs irradiated in the other regimes. Thus, the irradiation of eggs with a beam of accelerated protons in the Bragg peak in the FLASH/SPLASH modes is less damaging for healthy tissues and for the development of embryos and chicks on the cellular, anatomical, and physiological levels.

PurposeDetermination of the value of relative biological effectiveness (RBE) of heavy charged ions in vivo is an important task for their optimal use in particle radiotherapy. The aim of this study was to determine the RBE value of a beam of carbon ions with an energy of 450 MeV/nucleon in different regions of the Bragg curve in irradiation of mice at low, medium, and high doses in comparison with X-ray radiation.Materials and methodsSHK mice (n = 330) were irradiated in three regions of the Bragg curve in the dose range of 0-1.5 Gy for cytogenetic damage detection and at a dose of 6.5 Gy for determination of 30-day survival. For irradiation of mice in the Bragg peak, two widths of a spread-out Bragg peak (SOBP) were used: 10 mm (LET similar to 100 keV/mu m) and 30 mm (LET similar to 39 keV/mu m).ResultsThe RBE value was 0.8-0.9 before the Bragg peak (LET similar to 15 keV/mu m) and 0.8 after the peak (LET similar to 5 keV/mu m), and did not depend on the determination method, despite the differences in LET values. The RBE value determined by the micronucleus test was 1.1-1.7 for the 10-mm-wide SOBP and 1.0-1.3 for the 30-mm-wide SOBP, with the highest RBE value obtained in the low-dose region upon irradiation of mice in the 10-mm-wide Bragg peak. The RBE values in the high-dose region determined by the 30-day survival test lay in the range from 1.4 to 2.6 depending on the width of the Bragg peak and the chosen criterion for calculating the value. The RBE values in the 10-mm-wide Bragg peak (LET similar to 100 keV/mu m) were higher than those in the 30-mm-wide Bragg peak (LET similar to 39 keV/mu m) at all used criteria.ConclusionsThe present findings suggest that there is the complex relationship between LET and organism response to accelerated charged particle radiation, and the contribution of specific factors and mechanisms must be further considered.

The mitochondrial inhibitors rotenone and rhodamine 123 and the Janus Green B dye are being studied in order to develop pharmacological agents that cause mitochondrial dysfunction and apoptosis. Since mitochondrial dysfunction is associated with the production of reactive oxygen species, it seems relevant to compare the DNA damage induced by these substances to cells of ascitic Ehrlich carcinoma and murine lymphocytic leukemia P388 with exposure to a known inducer of reactive oxygen species, that is, ionizing (X-ray) radiation. The level of DNA damage was assessed using an alkaline version of the Comet assay. The level of DNA damage induced by rotenone was comparable to irradiation at a dose of 4 Gy in both cell types. Post-radiation incubation reduced the level of DNA damage, which indicates DNA repair. Treatment of Ehrlich’s ascitic carcinoma cells with rhodamine 123 followed by washing did not cause this increase; however, irradiation at a dose of 4 Gy in the presence of rhodamine 123 induced an increase in the level of DNA damage, which significantly decreased after 1 h incubation. It can be assumed that pretreatment of cells with rotenone and rhodamine 123, which impair the work of mitochondria, contributed to the preservation of the integrity of nuclear DNA in irradiated cells. Exposure to Janus Green B caused increased DNA damage and cell death. Based on the alkaline version of the Comet assay, damage induced by these compounds can be considered as single- and double-strand breaks and alkali-labile (apurine/apirimidine) sites in DNA.