FLASH irradiation does not induce lipid peroxidation in liposomes containing cholesterol and α-tocopherol

A decrease in lipid peroxidation (LPO) under higher dose rate irradiation could play a role in the FLASH sparing effect. Our objective was to explore how LPO induced at FLASH (> 100 Gy/s) and conventional (CONV, < 10 Gy/min) dose rates in synthetic phospholipid membranes depends on concentrations of cholesterol (Chol) and α-tocopherol (Toc). Solutions of phosphatidylcholine (PC) liposomes, either alone, with Chol or with a combination of Chol and Toc, were irradiated by FLASH and CONV electron beams. In addition, samples incorporating both Chol and Toc were tested under a range of gradually changing dose rates. LPO yield was assessed by oxygen consumption measurements and EPR spectrometry of spin probes (TEMPONE and mito-TEMPO). Regardless the cholesterol or tocopherol concentrations, LPO was considerably reduced when samples were irradiated at FLASH dose rates. Increasing Chol concentration led to a decrease in LPO yield upon irradiation, mostly likely due to the less oxidizing index of cholesterol compared with linoleic acid and increased stiffness of the membrane, hindering O 2 diffusion. Increasing Toc concentration led to an enhanced protection of the membrane from LPO. Increasing the average dose rate diminished the oxidative potential of the beam. EPR measurements demonstrated interaction of spin probes with membrane radicals in CONV, but not in FLASH. Irradiation at FLASH dose rates was able to inhibit LPO in PUFA-containing artificial PC membranes under all tested conditions, demonstrating that the FLASH beam creates less oxidizing damage to a phospholipid membrane than a conventional beam. • FLASH avoids lipid peroxidation in liposomes containing cholesterol and tocopherol • 7-DHC limits lipid peroxidation by reducing membrane damage from truncated lipids • Increasing dose rate lowers potential of radiation to oxidize phospholipid membrane • EPR measured negligible radical-spin probe interaction in the membrane during FLASH • FLASH exhibits less pro-ferroptotic impact than conventional radiotherapy