A Reactive Oxygen Species Approach For Absolute Radiation Dosimetry

Stereotactic Ablative Radiosurgery (SABR) achieves elevated local control through the creation of DNA damage and reactive oxygen species (ROS). Hydrogen peroxide is a pivotal ROS created during radiotherapy that alters aspects of cell physiology by reacting with biomolecules, creating secondary ROS, and generating oxidative stress. This suggests H2O2 production/removal is significant in the differential survival of normal and tumor cells, making it evident that manipulating H202 levels during radiation therapy could further maximize this difference. We hypothesized that the hypersensitivity response and therapeutic gain observed in SABR is mediated, at least in part, through the increased production of H202. To investigate this, we developed a fluorometric technique to measure SABR’s potential to generate H202. Materials/Methods Hydrogen peroxide was quantified using the Resorufin H2O2 Assay following the manufacturer’s guidelines. To measure H2O2 levels after exposure, 100 μl of Ultra Trace water was exposed in a 10 x 10 cm2 field, 100 cm SSD with 10XFFF radiation at dMax at doses between 0.01 – 200 Gy. Samples were also exposed to Ir-192 HDR, CT scans, and various energies of photons and electrons. Fluorescence was measured using a commercially available fluorometer. The absolute [H2O2] was calculated using a standard curve of known H2O2 concentrations. Lastly, H2O2 was confirmed as our signal’s source by incubating matched samples with 100 U of Catalase prior to analysis. Results A linear relationship between Resorufin fluorescence and dose exists within a range 0.05 - 200Gy in 10X FFF. Likewise, [H2O2] was linearly related to dose with values between 0.01μM – 24.9μM, yielding 0.651μM/Gy, and 0.675μM/Gy (R2 = 0.99) within ranges of 0.05-20Gy, and 0.05-200Gy, respectively. Preincubation with catalase returned fluorescence to background levels, suggesting H2O2 is the molecule we are measuring. These results suggest this assay is sensitive and can detect exposures over a broad dose range. Similar results we observed for other sources of ionizing radiation. Therapeutic strategies in radiation oncology such as SABR and Flash therapy improve local control; however, the mechanism(s) responsible remains elusive. Normal and tumor cells exhibit differences in their redox biologies that might partially account for this. We suggest higher levels of ROS, principally H2O2, alters different aspects of cell biology in normal and tumor cells including apoptosis, metabolism, and the formation of gas emboli. To investigate this, we developed a sensitive fluorometric technique to measure H2O2 produced during irradiation. These results show our technique is capable of measuring exposure for a broad range of photon and electron energies, dose and dose rates, suggesting this dosimetry method more accurate than conventional methods during SABR and Flash therapy. Further research is warranted.