RADIATION-INDUCED DNA-DAMAGE IN TUMORS AND NORMAL-TISSUES .2. INFLUENCE OF DOSE, RESIDUAL DNA-DAMAGE AND PHYSIOLOGICAL FACTORS IN OXYGENATED CELLS

Detection and quantification of hypoxic cells in solid tumors is impor tant for many experimental and clinical situations. Several laboratori es, including ours, have suggested that assays which measure radiation -induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elutio n assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must no t be formed to any great extent in irradiated oxygenated cells, and th e formation and repair of strand breaks and DPCs in oxygenated cells m ust not be modified appreciably by physiological factors (e.g. tempera ture, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In o ne set of experiments, oxygenated 9L cells in tissue culture, subcutan eous 9L tumors and rat cerebella were irradiated with doses of 15 or 5 0 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated e xponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of st rand breaks or the formation of DPCs in oxygenated 9L cells. These res ults support the use of this DNA damage assay for the detection and qu antification of hypoxic cells in solid tumors.