To establish the optimal experimental conditions for the use of the micronuclei (MN) test to determine the level of chromosomal damage induced by ionising radiation (IR) exposure in lymphoblastoid cell lines, a time-course study was performed comparing a normal and an ataxia telangiectasia (AT) cell line, the latter being characterised by an extreme radiation sensitivity. Several parameters were analysed: the use of cytochalasin-B (Cyt-B) to quantify MN, the optimum fixation time to measure radiation-induced MN, the most appropriate treatment dose of IR to distinguish between the normal and the radiosensitive cells and the cell-cycle distribution after irradiation. The results obtained showed that the spontaneous as well as the radiation-induced levels of MN were significantly higher in the AT cell compared to the normal cells (P < 0.001 and P = 0.005, respectively). In both cell types the number of radiation-induced MN were lower in the cultures without Cyt-B than those with Cyt-B (P < 0.001), with the AT cells being distinguished in terms of IR-induced MN from the normal cells only with the addition of Cyt-B. The level of MN formation was independent of the dose of Cyt-B used (3 or 6 microg/ml). The optimum time for radiation-induced MN measured was found to be between 48 and 72 h post-irradiation, with 2 and 4 Gy exposures inducing similar levels of MN. However, as the higher dose caused a greater delay in the cell-cycle, treatment with 2 Gy with MN measurement at 48 h in the presence of 3 microg/ml Cyt-B were chosen as the optimum experimental conditions. This choice was validated using two additional normal and AT cell lines. In conclusion, our results show that the use of Cyt-B increases the sensitivity of the MN test for comparing differences in radiosensitivity between lymphoblastoid cell lines.
Copyright 2002 Elsevier Science B.V.