DETECTION OF HYPOXIC CELLS IN A C3H MOUSE MAMMARY-CARCINOMA USING THECOMET ASSAY

The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours impl anted into the feet of female CDF1 mice. Tumours were either clamped b efore irradiation or mice were allowed to breath air, 100% oxygen, car bogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiat ion and individual tumour cells were analysed for DNA single-strand br eaks. Hypoxic cells were defined as those cells with approximately thr ee times fewer single-strand breaks than aerobic cells. Radiobiologica l hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay. Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic frac tion from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r(2) = 0.994). In th e SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured us ing the comet assay and hypoxic fraction measured in the same tumour c ells using the paired survival curve assay (slope = 1.20 with 95% conf idence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tum our types.