In vitro oxygen concentration alters reactive oxygen species yields with minor plasmid DNA damage change at ultra-high-dose rate

Abstract Objective Ultra-high-dose rate (UHDR) radiotherapy has become a large area of research due to observed normal tissue sparing without sacrificing tumour control, termed the FLASH effect. The purpose of this study was to compare reactive oxygen species (ROS) production and DNA damage across various O2 levels at UHDR and conventional dose rates (CDR) in solutions without repair enzymes and radical scavengers. Methods Solution assays of both ROS and DNA damage assessed dose rate and oxygen dependent (0%-20% O2) changes between UHDR and CDR from an IntraOp Mobetron. For ROS reporters Amplex UltraRed (H2O2), and CellROX Deep Red (non-H2O2) were quantified via intensity per unit dose. DNA damage assayed plasmid pBR322 gel electrophoresis, to differentiate both single (SSB) and double strand breaks (DSB). Results For ROS assays, a significant reduction was noted from CDR to UHDR across all measured oxygen levels. The generation of H2O2 decreased when departing from physiologically relevant oxygen levels (1%-5%), with generation 30%-40% lower at UHDR. The DNA damage assay showed no trends in the SSB or DSB values with O2. Conclusion Examination of trends between ROS and DNA damage from factors such as oxygen can help elucidate FLASH mechanisms. The H2O2 yield has maximum yield at physiological oxygenation levels (1%-5%), and UHDR further diminishes yield. In DNA damage no trend was observed. It is possible that these mechanisms have underlying effects on the FLASH effect in vivo. Advances in knowledge This study is the first to directly compare radiation chemistry differences caused by UHDR to biologically relevant DNA damage in identical solutions.