Radiochemical Oxygen Depletion Depends on Chemical Environment and Dose-Rate: Implications for FLASH Effect

Abstract

FLASH (dose rates > 40 Gy/s) radiotherapy (RT) protects normal tissues from radiation damage, compared with conventional RT (∼Gy/min). Radiation-chemical oxygen depletion (ROD) has been the leading and most-studied mechanism to explain FLASH. Previous studies have reported low ROD values of ∼0.35 µM/Gy, however, they do not accurately model (utilizing water or oxidized substrates such as albumin) the intracellular environment. It has been proposed that the intracellular environment may support higher ROD values due to its specific chemical makeup. As chemical reducing agents, which play a critical role in the cell, may promote ROD, we measured ROD for several well-known cellular reducing agents. Precision polarographic oxygen sensors were used to measure ROD over a ∼100-0 µM oxygen concentration range in solutions containing various types of intracellular reducing agents, either alone or combined with glycerol (1M; to simulate the intracellular hydroxyl-radical-scavenging capacity). A Cs irradiator and a research proton beamline were used for radiation beam generation over a 10,000-fold (0.0085 to 100 Gy/s) dose rate range. Reagents were grouped generally into primary free-radical scavengers (glycerol & lysozyme) and reducing agents (cysteamine, cysteine, glutathione, dextrin-conjugated linoleic acid, NAD(P)H, ascorbate and uric acid). Reducing agents at 1 mM significantly altered ROD values. ROD, at 0.1 Gy/s varied from greater than 1 µM/Gy for NADPH to less than 0.1 µM/Gy for uric acid. Even higher RODs were found at lower dose rates and/or higher reducing agent concentrations. Although most greatly increased ROD, ascorbate and uric acid decreased ROD and additionally imposed an oxygen dependence of ROD at low oxygen concentrations. Interestingly, all agents having high rates of ROD at low dose rates show a decrease at FLASH dose rates and this seems to be a proportional response (i.e., the greater the ROD at low dose rate, the greater the inhibition by FLASH). At FLASH dose rates our data for a simple mixture of glycerol and glutathione showed the same trend as recent studies that, in general, FLASH had ∼25% lower ROD rates than conventional demonstrating agreement between polarographic sensors and optical phosphorescence decay methods. ROD was greatly augmented by some intracellular reducing agents but others effectively reversed this effect. Ascorbate had its greatest impact at low oxygen concentrations. ROD decreased with increasing dose rate and in all cases was less than 1 µM/Gy at 100 Gy/s, potentially posing a fundamental limit of max ROD for FLASH.