Background and Aims

The Monte Carlo track structure code TRAX has been recently extended into the pre-chemical and heterogeneous chemical stages of radiation damage, in a water target up to 1μs after irradiation (“TRAX-CHEM”, Boscolo et al., 2018). Notwithstanding that, to understand the fate and final biomolecular effects of the species produced, a further extension is needed. In this regard, the “homogeneous” biochemical stage (up to ~ s or longer), including target biomolecules, is under development.

Methods

In contrast to the discrete, step-by-step description of all species in the early track evolution, the new implementation uses a continuous representation based on concentration distributions. Radicals and molecules of neighbouring tracks diffuse and react with each other and with the biological environment, influencing their chemical yields and generating new products. Computationally, after importing the spatial distributions of all chemical species from TRAX-CHEM, the set of reaction-diffusion equations is solved numerically up to longer time points.

Results

The developing model has been validated against TRAX-CHEM for the set of reactions describing a pure water environment (Figure 1), with an improvement in the computational time by more than three orders of magnitude. In addition, the optimal time point for a smooth transition between the two approaches has been found to lie around 500ns, by monitoring that the differences in the total amount of species produced at 1μs do not exceed 3-5% and the spatial distributions agree closely.

Conclusions

The new implementation is able to follow the effects of different particle qualities, characterised by energies and LET values within the therapeutic regime, up to the time scale of a second. The next step is to benchmark the predictions with experimental data, e.g. under ultra-high dose rate (“FLASH”) radiotherapy. Moreover, biomolecules and other reaction partners such as DNA nucleotides, lipids and enzymatic antioxidants will be included in the simulation.

Background and Aims

To investigate the impact of temporal beam structure on physico-chemical events following exposure to ultra-high (UHDR) and conventional dose rates, non-buffered water and pBR322 plasmids were irradiated using: eRT6/Oriatron/CHUV 5.5MeV electron beam at 0.1Gy/s (conventional) and ≥555Gy/s (UHDR); Gantry 1/COMET cyclotron/PSI 235MeV proton beam in transmission mode at 1Gy/s (conventional) and 1400Gy/s (UHDR); and CLEAR/CERN 220MeV electron beam at 0.2Gy/s (conventional) and 10⁹ Gy/s (UHDR).

Methods

G°(H₂O₂) were estimated with scavenging methods while G(H₂O₂) were measured minutes after water irradiation. DNA damage was quantified after plasmid irradiation in presence or absence of hydroxyl radicals’ scavenger (DMSO) by gel electrophoresis (AGE).

Results

When CONV and UHDR-irradiation were compared, similar G°(H₂O₂) were found with electron, proton and VHEE. However, electron produced the highest primary yield of H₂O₂ (0.8±0.01 and 0.75±0.02 molecules/100eV), that was reduced with proton (0.66±0.02 and 0.67±0.01 molecules/100eV) and even lower with VHEE (0.56±0.02 and 0.52±0.02 molecules/100eV). G(H₂O₂) was significantly lower after UHDR irradiation compared to CONV-RT. The reduction was similar for all 3 beams (31% for 5.5MeV electrons; 35% for protons and 37% for VHEE). No difference in plasmid DNA damage was measured after UHDR vs CONV for any of the beams when plasmids were irradiated in 21%O₂. Investigation of scavenger’s effect is ongoing.

Conclusions

These experiments are the first systematic investigations involving various beams able to operate at UHDR with different temporal structure. The results show that initial physico-chemistry events post-irradiation are dose rate independent, whereas UHDR irradiation lowers G(H₂O₂) values at later time point, suggesting modification of recombination/diffusion processes at UHDR. This does not impact DNA damages in plasmids but needs to be investigated in biological systems.

Acknowledgement: The study is funded by FNS Synergia grant (FNS CRS II5_186369).

Background and Aims

To investigate the role of oxygen in radio-induced damage to unsaturated lipids, UPLC coupled to tandem mass spectrometry (UPLC/ESI-MS-MS) was used for analysis of twenty hydroxy- and oxo-derivatives (oxylipins) of docosahexaenoic (HDOHE), arachidonic (HETE, Oxo-ETE, LTB4, 8-epiPGF2α) and eicosapentaenoic acids (HEPE) in normal cells exposed to FLASH vs. conventional dose-rate irradiation (CONV) in close equilibrium with 20% vs. 1% O₂ at 37°C.

Methods

Human retinal pigment epithelial RPE1-hTERT cells were grown to confluence at low serum in equilibrium with air-5%CO₂ or incubated overnight with 1%O₂-5%CO₂-94%N₂ prior to irradiation. Hermetically sealed flasks were given 10, 20 or 30 Gy FLASH (7 MeV, 3 Gy/pulse at 6.7 ms interval, 8x10⁵ Gy/s, duty time 13.3-193 ms) vs. CONV irradiation (10 Gy/min). Cells were collected at exactly 5 min following radiation and stored in liquid nitrogen. Fatty acids released upon alkaline hydrolysis of the cell pellets were submitted to UPLC/ESI-MS-MS.

Results

In cells irradiated in air, 10 Gy FLASH or CONV eliminated 50% of endogenous oxylipins in the Oxo-ETE series, followed by dose-dependent neoformation of the oxylipin in the CONV mode only. The same effect was observed in other oxylipins, yet to a smaller extent. In all series FLASH was unable to induce neoformation of oxylipins, in such a way that the level of oxylipins after FLASH remained 20-50% below that in controls and CONV-irradiated cells. Hypoxia elicited a large increase in endogenous 4-HDOHE, 5-HEPE and 8-epiPGF2α with suppression of the advantage of FLASH in these series and in 15-HETE.

Conclusions

Radiation-induced formation of oxylipins was quenched in the FLASH mode independently of [O₂], putatively via bimolecular recombination of primary radical species. Hypoxia profoundly altered the metabolism of four oxylipins and suppressed the differential CONV vs. FLASH response in these phospholipids.

Acknowledgments Financial support to this study came from Inserm grant No. 19CP122-00

Background and Aims

The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV) ionizing radiation (IR) dose-rate are not known. However, attenuated DNA damage and transient oxygen depletion are among a number of proposed models. We tested whether FLASH-IR under hypoxic conditions (<2% O₂) attenuates detection of genome-wide translocations relative to CONV dose rates and whether any differences identified revert under normoxic (21% O₂) conditions.

Methods

We employed high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), using S. pyogenes and S. aureus Cas9 “bait” DNA double strand breaks (DSBs), to measure differences in proximal repair and their translocation to “prey” genome-wide DSBs generated by CONV (0.13Gy/s) and FLASH (2.10³-5.10⁶ Gy/s) dose rates, under different oxygen tensions, and at varying (2-20Gy) IR doses.

Results

IR exposure in HEK293T cells at normoxic (21% O₂) conditions increased both the proportion and total number of translocations from the junctions recovered but were highly similar between CONV-RT and FLASH-RT dose-rates. Although increased proportions of translocations were observed as cells were acutely transitioned to either physioxic (4%) or hypoxic (<2%) conditions alone, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures, which were predominantly increased in microhomology utilization.

Conclusions

We conclude that irrespective of oxygen tension, ultra-high dose rate IR produces translocations and junction structures at levels that are indistinguishable from conventional dose rates.

Background and Aims

FLASH irradiation (FLASH-IR) is efficient in tumor control while reducing radiotoxicity to normal tissues. However, the mechanism of this FLASH effect is still unclear and limited data have been reported on this effect on liver. This study aims to compare the radiobiological response of liver upon FLASH-IR or conventional dose-rate IR (CONV-IR).

Methods

Female FvB mice were randomly assigned into three groups (the non-irradiated control group, the CONV-IR group and the FLASH-IR group. Liver injury was evaluated by H&E staining, while serum level of ALT and AST was determined by ELISA. DNA damage and cell apoptosis in liver was analyzed by γH2AX and TUNEL staining, and neutrophils and macrophages in liver was analyzed by IHC staining of MPO and F4/80, respectively. Inflammatory state was evaluated by IHC staining of cytokines, TGFβ1 and STING, and liver fibrosis was evaluated by Sirius red and αSMA staining.

Results

The serum ALT level in FLASH-IR was lower than CONV-IR. Besides, the γH2AX foci in both IR groups were higher than control, while that in FLASH-IR was lower than CONV-IR. Compared with control, the level of TNF-α, IFN-γ and IL-6 was increased in CONV-IR with IL-10 decreased, while the level of IFN-γ and IL-6 was also increased in FLASH-IR. Besides, the level of TNF-α, IFN-γ and IL-6 in FLASH-IR was lower than CONV-IR. Moreover, liver steatosis and apoptosis were alleviated in FLASH-IR compared with CONV-IR. Furthermore, the number of TGF-β1+ cells, STING+ cells and MPO+ neutrophils was increased in CONV-IR but not FLASH-IR, while no difference in liver fibrosis and macrophage infiltration was found.

Conclusions

FLASH-IR showed reduced liver injury, which may be due to reduced inflammatory responses and MPO+ neutrophils infiltration. These findings suggest that FLASH-IR may be an effective strategy for improving the therapeutic index of liver radiotherapy.