Morimoto K, Sato-Mizuno M, Koizumi A
Mutat Res. 1985 Nov-Dec;152(2-3):187-96. doi: 10.1016/0027-5107(85)90060-0.
Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.
已开展实验，使用人全血培养物来确定采样时间以及固定前5-溴脱氧尿苷（BrdUrd）处理持续时间对暴露于丝裂霉素C（MMC）后姐妹染色单体交换（SCE）频率的影响。细胞在G1期用3×10⁻⁶ M MMC脉冲处理1小时，然后在用植物血凝素（PHA）刺激培养物后，每隔4小时取样一次，直至88小时。结果表明，这种MMC处理每个细胞周期诱导5 - 6小时的增殖延迟，并且SCE频率首先随固定时间增加，在68小时达到峰值，然后下降。当细胞用MMC进行类似处理，但随后在72小时固定培养物之前用BrdUrd处理不同时间时，SCE频率随BrdUrd孵育时间的增加而显著增加。这些数据表明，在经诱变剂处理的培养物中，细胞周期时间相对较长的淋巴细胞显示出较高的平均SCE频率。在随后的实验中，细胞在0、24或48小时用递增剂量的MMC或4-硝基喹啉1-氧化物（4NQO）处理1小时，并在PHA刺激后72小时固定。结果表明，可以最有效地产生SCE的这些试剂的最佳处理时间对于MMC和4NQO是不同的，并且剂量反应曲线在非常高的剂量时倾向于“向下弯曲”；也就是说，用非常高剂量处理诱导的SCE数量比预期的少。然而，用非常高剂量进行类似处理的细胞在84小时取样时显示出较高的、预期的SCE频率，但在96小时固定时再次具有低于预期的SCE频率。结果表明存在一个最佳采样时间，在该时间可以观察到诱变剂暴露后SCE频率的最大增加，并且强烈表明剂量越高最佳采样时间越晚。由于在各种处理和采样时间后获得的剂量反应曲线明显畸形，似乎有必要在测试方案中纳入额外固定时间点，以避免假阴性或确认可能的阳性结果。
