Background and Aims

Ultra-high dose rate (UHDR) irradiation produces the FLASH effect (anti-tumor effect without normal tissue toxicity) at average dose rates above 100 Gy/s. Two physico-chemical scenarios were proposed as a mechanistic basis for the FLASH effect following water radiolysis: 1. Altered radical-radical reactions and 2. Depletion of O₂ by free radicals. To investigate these questions, we determined primary radiolytic yields (G°-values) of hydrogen peroxide and hydroxyl radicals. G(H₂O₂) was also determined in low O₂ condition (1%), intermediate levels (4%) and atmospheric conditions (21%) following homogenous phase. O₂ depletion was also measured.

Methods

Scavenging methods were used to estimate radiolytic yields of H₂O₂ in water samples. Hydroxyl radicals production was estimated using EPR spin trapping with DMPO. O₂ measurements were performed using OxyLite probe.

Results

When UHDR and CONV-irradiation were compared, similar primary yields of H₂O₂ were found and EPR measurements suggested no differences in HO· production. However, G(H₂O₂) was significantly lower after irradiation at UHDR in samples equilibrated at 4%. O₂ measurements resulted in similar but low depletion with both modalities at intermediate and atmospheric O₂ conditions, whereas at low O₂ level, oxygen depletion was lower at UHDR.

Conclusions

These observations suggest that initial radiation chemistry is similar in both modalities: Similar yields of radicals ‘‘escaping’ ’track recombination after ionization. However, irradiation at UHDR produces less H₂O₂ at intermediate O₂ levels following initial chemistry events, supporting occurrence of scenario 1. O₂ depletion hypothesis is not favored by results obtained in pure water.

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

Background and Aims

In FLASH radiotherapy (RT), a protective effect of healthy tissue was observed, while tumor control remains comparable to conventional RT. One possible explanation is the oxygen depletion hypothesis, in which radiolysis of water/cytoplasma causes the production of radicals that then react with O2 dissolved in water. This would cause a reduction in O2, which results in a hypoxic target and thus a radio-protective effect. In a previous study (Jansen et al. 2021), we measured O2 depletion using an optical sensor in sealed water phantoms during irradiation at high dose rates (<300 Gy/s) for protons, carbon ions and photons.

Methods

In the study presented here, this experiment was conducted further to ultra-high dose rates (10^9 Gy/s) with protons at DRACO and electrons at ELBE, where also the impact of different pulse structures on O2 depletion in water was tested. In addition, various settings were tested in order to irradiate zebrafish embryos with FLASH while simultaneously measuring O2.

Results

We were able to confirm the results of our previous study even at ultra-high dose rates and with electrons. Furthermore, it was possible to measure O2 depletion during zebrafish embryo irradiation making a simultaneous study of biological response and O2 depletion possible.

Conclusions

Not enough O2 was depleted at clinical doses to explain a FLASH effect based on radiation-induced hypoxia. The amount of O2 depleted per dose depends on dose rate, and higher dose rates deplete slightly less O2. The experimental set up allows for future joint experiments of biological samples and oxygen monitoring.

Background and Aims

Convincing evidence has been provided that lowering the mean dose rate below 30 Gy/s (Montay-Gruel et al. Radiother Oncol 124: 365-369, 2017) or increasing the oxygen tension through carbogen breathing (Montay-Gruel et al. PNAS 116: 10943-10951, 2019) can reduce (if not eliminate) the sparing effect of FLASH in the mouse brain. Three theoretical models have since been proposed to account for the dependence of FLASH on oxygen, namely, (i) Radiation-induced transient oxygen depletion (TOD), (ii) Cell-specific differences in the ability to detoxify and/or recover from injury caused by reactive oxygen species, (iii) Self-annihilation of radicals by bimolecular recombination.

Methods

These theoretical models were confronted to experimental evidence relating to physical chemistry and the response of biological systems to ultrahigh dose rate irradiation in light of the chemical reactions underlying the radiosensitizing effect of O₂ from immediate, early processes to late complications.

Results

The radiosensitizing effect of oxygen has long been known to stem from peroxyl radicals (ROO^•). Peroxyl radicals areformed by the addition of O₂ in its fundamental, triplet state to short-lived carbon-centered radicals (R^•) generated upon H^• atom abstraction from aliphatic, unsaturated or conjugated substrates (RH).

Conclusions

Any attempt at deciphering the physical-chemical processes that underpin the FLASH effect must take into consideration the fate of these radicals, and in particular the probability of radical recombination. The radiation dose rate emerges as the most important factor. We propose testable procedures to investigate the mechanisms that underly the FLASH effect in normal cells, and differential tumor vs. normal cells responses.

Background and Aims

Radiotherapy at ultrahigh dose rates (FLASH, >40Gy/sec) compared to conventional radiotherapy (CR; <1 Gy/sec) has been shown to provide an advantage by selective protection of normal versus tumor tissue. Depletion of molecular oxygen causing radioprotection has been proposed to underpin the FLASH effect, however no experimental data have been presented to test this hypothesis. Our goal was to assess the oxygen dynamics during and following proton FLASH versus CR.

Methods

Measurements of oxygen were performed in vitro (solutions in sealed vials) and in vivo in mice (healthy leg muscle and tumors) using the phosphorescence quenching method with probe Oxyphor PtG4 at rates reaching 3 measurements/millisecond during proton irradiation delivered with CR or FLASH dose rates.

Results

In closed systems in vitro the rate of oxygen depletion is oxygen-independent for both FLASH and CR when [O₂]>~70 μM. The g-values are lower for FLASH than for CR by ~15%. In vivo oxygen is depleted upon FLASH with rates that correlate with baseline pO₂ (partial pressure of oxygen) values throughout the entire pO₂ range: at 30-40 mmHg (normal tissue) a 30 Gy FLASH (100 Gy/s) results in ΔpO₂ of -8 mmHg, while at 5-7 mmHg (tumor) ΔpO₂ is only -1 mmHg.

Conclusions

This study features the first demonstration of the phosphorescence quenching oximetry at ultrafast rates and in the presence of proton radiation. Oxygen depletion by FLASH in vitro was found to be smaller than by CR, and appears to correlate with baseline pO₂ values, being greater for normal tissue than for tumors.

Background and Aims

Several models assume that delivering radiation at ultra-high dose rates (UHDR) reduces the yields of reactive oxygen species (ROS). Montay-Gruel et al [1] measured a significant decrease of Hydrogen Peroxide (H₂O₂) after UHDR irradiation compared to conventional dose-rate irradiation (CONV) with electrons. This study aims to verify this assumption in proton beams. We study the UHDR radiation chemistry by the measurement of H₂O₂ produced by the water radiolysis. Fricke dosimeter is used as dose control and as ROS sensor as well.

Methods

Using ARRONAX facility, we produced proton beams (68MeV) ranging from low (0.25Gy/s, 100Hz, pulse dose rate=6.3Gy/s) to ultra-high dose rates (7500Gy/s, single pulse). Doses ranging from 5Gy to 80Gy were delivered to 1.5ml Eppendorf tubes filled with water. In this investigation, Fricke dosimetry [2] is used to verify the dose for both irradiation modes. The second step is the determination of H₂O₂ concentrations after irradiation with the Ghormley triiodide method [3].

Results

In this work, we have brought the evidence of the Flash effect by the H₂O₂ measurement in the two cases: (i) For CONV irradiations, we determined a H₂O₂ chemical yield close to the literature at 0.9 10^-7 mol.J^-1 [4], (ii) For UHDR irradiations, this yield is measured to a significant lower value. Observed Fricke values are the same for both modes.

Conclusions

We have revealed a radio-chemical FLASH effect for the proton beam by the H₂O₂ measurement as radiolytic species during the irradiation of water. Further studies will be performed to determine the beam time structure effect.

Background and Aims

In our work we thought to compare the effects of conventional (CONV) vs ultra-high dose rate (UHDR) by quantifying DNA strand breaks (SB) after irradiation of plasmid-DNA (pBR322) under various oxygen concentrations.

Methods

Supercoiled pBR322 was irradiated dry or in water using a 6 MeV FLASH-validated electrons beam, with increasing doses (1-100 Gy) and dose per pulse (0.01 Gy/s (CONV), 5.0*10² to 5.6*10⁶ Gy/s (UHDR)) and at atmospheric (21%), physoxic (4%) and hypoxic (0.5%) oxygen level. The increase of relaxed (R) and linear (L) plasmid forms after irradiation was quantified by agarose gel electrophoresis and used to compute single and double SB yields.

Results

Dry, atmospheric conditions cause similar yields of SB in CONV and UHDR. Aqueous conditions shows higher SB yields as expected. Physoxia induces radioprotection compare to atmospheric condition: 50% of R at 10 Gy (4% O₂) vs 2 Gy (21% O₂), but no difference relative to dose rate. Hypoxia revealed higher SB yields than physoxia in CONV (50% of R at 6 Gy) but 2x less SB in UHDR for doses >30 Gy (see figure for L).

Conclusions

First results in dry condition suggest that direct effects are not involved in FLASH. In aqueous condition, 4% oxygen mimicking healthy tissues shows no difference between UHDR and CONV, while 0.5% oxygen mimicking tumors shows less damages in UHDR. These results are opposite to the preclinical results showing the FLASH effect. Thus, plasmid irradiation might be useful to understand DNA damage at UHDR but seems barely relevant to investigate the FLASH effect at the biological level.

Background and Aims

Transient changes in oxygen tension in tissues taking place during FLASH radiotherapy may explain its biological effects. However, because the kinetics of oxygen depletion and recovery occur on a very short timescale, it is challenging to measure these effects in vivo using existing methods. Here we developed a real-time optical oximetry system with millisecond temporal resolution to elucidate early radiochemistry under irradiation.

Methods

Oxygen measurements were performed in vitro using the phosphorescence quenching method and a water-soluble molecular nanoprobe (Fig. 1). An epifluorescence fiber-coupled system was designed and built. The system was validated using a standard dissolved oxygen meter. The changes in oxygen per unit dose (G-value) were quantified in response to irradiation by 320 kVp x-ray and 16 MeV electron beam at dose rates ranging from 0.04 Gy/s to 100 Gy/s.

Results

Transient oxygen depletion of phosphate buffer solution under standard kV X-ray irradiation was continuously measured every millisecond (Fig. 2). The samples at normoxia with oxygen concentration of 150–240 µM had constant G-value of 0.54 uM/Gy, however hypoxic samples (15 µM and below) had significantly lower G-values (Fig. 3).

Conclusions

Our observations suggest that oxygen depletion rate decreases under hypoxia, with the measured data being a good fit to Michaelis-Menten kinetics. Future works will measure oxygen depletion kinetics of biomimetic lipid emulsions and in vivo mice under irradiation. We anticipate that the proposed method will capture crucial millisecond-order oxygen change after individual e-beam pulses.