HYPOXIC FRACTIONS MEASURED IN MURINE TUMORS AND NORMAL-TISSUES USING THE COMET ASSAY

Purpose: To apply the alkaline comet assay to the detection of radiobi ologically hypoxic cells in solid tumors and normal tissues of mice, a nd to examine the influence of strand break repair on the oxygen enhan cement ratio measured using the alkaline comet assay. Methods and Mate rials: In previous studies, we found that hypoxic fraction in squamous cell carcinomas growing in C3H mice could be reliably and easily meas ured using the alkaline comet assay. The comet assay applies fluoresce nce microscopy acid image analysis to examine patterns of migration of deoxyribonucleic acid from individual cells embedded in agarose and e xposed to an electric field. This method has sufficient resolution to detect subpopulations of hypoxic cells which show about 3 X fewer stra nd breaks than aerobic cells after irradiation. Results: Fast rejoinin g kinetics in vitro are comparable to those measured in vivo, and rejo ining of strand breaks in hypoxic tumor cells occurs at a similar rate as rejoining in aerobic cells. Little residual damage was detectable using the comet assay in tumors 4-24 h following 15 Gy, allowing repea t measurements to be performed. Bone marrow and testis, but not liver, spleen, or jejunum contained a small fraction of hypoxic cells when m ice breathed 10% oxygen during irradiation. Conclusion: The comet assa y confirms that some normal tissues may border on hypoxia. Rejoining o f strand breaks occurs rapidly in both oxic and hypoxic cells so that the oxygen enhancement ratio remains relatively constant with time aft er irradiation. Interestingly, a smaller oxygen enhancement ratio was observed in tumors than was expected, probably as a result of the pres ence of acutely hypoxic cells.