Oxygen Monitoring in Model Solutions and In Vivo in Mice During Proton Irradiation at Conventional and FLASH Dose Rates

Abstract

FLASH is a high-dose-rate form of radiation therapy that has the reported ability, compared with conventional dose rates, to spare normal tissues while being equipotent in tumor control, thereby increasing the therapeutic ratio. The mechanism underlying this normal tissue sparing effect is currently unknown, however one possibility is radiochemical oxygen depletion (ROD) during dose delivery in tissue at FLASH dose rates. In order to investigate this possibility, we used the phosphorescence quenching method to measure oxygen partial pressure before, during and after proton radiation delivery in model solutions and in normal muscle and sarcoma tumors in mice, at both conventional (Conv) (∼0.5 Gy/s) and FLASH (∼100 Gy/s) dose rates. Radiation dosimetry was determined by Advanced Markus Chamber and EBT-XL film. For solutions contained in sealed glass vials, phosphorescent probe Oxyphor PtG4 (1 μM) was dissolved in a buffer (10 mM HEPES) containing glycerol (1 M), glucose (5 mM) and glutathione (5 mM), designed to mimic the reducing and free radical-scavenging nature of the intracellular environment. In vivo oxygen measurements were performed 24 h after injection of PtG4 into the interstitial space of either normal thigh muscle or intra-muscular sarcoma tumors in mice. The “g-value” for ROD is reported in mmHg/Gy, which represents a slight modification of the more standard chemical definition (μM/Gy). In solutions, proton irradiation at conventional dose rates resulted in a g-value for ROD of up to 0.55 mmHg/Gy, consistent with earlier studies using X or gamma rays. At FLASH dose rates, the g-value for ROD was ∼25% lower, 0.37 mmHg/Gy. pO2 levels were stable after each dose delivery. For normal muscle in vivo, oxygen depletion during irradiation was counterbalanced by resupply from the vasculature. This process was fast enough to maintain tissue pO2 virtually unchanged at Conv dose rates. However, during FLASH irradiation there was a stepwise decrease in pO2 (g-value ∼0.28 mmHg/Gy), followed by a rebound to the initial level after ∼8 s. The g-values were smaller and recovery times longer in tumor tissue when compared to muscle and may be related to the lower initial endogenous pO2 levels in the former. Considering that the FLASH effect is seen in vivo even at doses as low as 10 Gy, it is difficult to reconcile the amount of protection seen by oxygen depletion alone. However, the phosphorescence probe in our experiments was confined to the extracellular space, and it remains possible that intracellular oxygen depletion was greater than observed herein. In cell-mimicking solutions the oxygen depletion g-vales were indeed significantly higher than observed in vivo.

Mean dose rate in ultra-high dose rate electron irradiation is a significant predictor for O2 consumption and H2O2 yield

“…The use of BSA as a dominant protein provides a first order match to in vivo testing, where albumin is the most dominant protein in the plasma. Still, it is possible, and perhaps likely that further interactions with the protein and lipid milieu present in vivo are even more complex than seen here (Van Slyke et al 2022 ). Intermediate steps towards in vitro or in vivo studies could utilize protein mixtures that approach in vivo .…”

“…The implications of these findings are significant to understanding the underlying mechanisms of the FLASH effect in addition to the parametric considerations of UHDR delivery. Hydroxyl radicals formed through water radiolysis play a significant role in radiation-induced DNA damage pathways (Blain et al 2022 , Van Slyke et al 2022 ). Interactions of these hydroxyl radicals with DNA can cause single- and double-strand breaks, which may lead to cellular and tissue damage.…”

“…Previous in vitro and in vivo studies using electron (Cao et al 2021 ) and proton (El Khatib et al 2022 , Van Slyke et al 2022 ) beamlines have shown a reduction in the oxygen partial pressure consumed (ΔpO 2 ) per-Gy of dose, for UHDR compared to conventional irradiations. In both studies, in vitro experiments resulted in lower consumption rates (O 2 consumed per unit dose) with UHDR compared to conventional.…”

Proton FLASH effects on mouse skin at different oxygen tensions

“…In vitro mammalian cell survival assays that observed a positive FLASH effect with <1% rather than 26% oxygen in the medium also implied the critical role of oxygen (Nias et al 1969, Berry and Stedeford 1972, Adrian et al 2020. However, the oxygen depletion g value for both electron and proton FLASH irradiation was reported to be lower than conventional irradiation in solution in vitro (Cao et al 2021, El Khatib et al 2022, Van Slyke et al 2022. The theory of ROD as the dominant mechanism of the in vivo FLASH effect remains controversial.…”

Comet Assay Profiling of FLASH-Induced Damage: Mechanistic Insights into the Effects of FLASH Irradiation

“…Recent studies measuring oxygen in solutions all indicate that more oxygen is depleted during CONV than FLASH exposures [ 55 , 56 , 57 ]. While measurements in vivo show a measurable change in oxygen content only during FLASH exposure, the consumption of oxygen per Gy is generally considered as too low to have a biological impact that would (solely) explain the FLASH effect [ 55 , 56 , 57 ].…”