Henderson L, Jones E, Brooks T, Chételat A, Ciliutti P, Freemantle M, Howard C A, Mackay J, Phillips B, Riley S, Roberts C, Wotton A K, van de Waart E J
Environmental Safety Laboratory, Unilever Research, Colworth House, Sharnbrook, Beds, UK.
Mutagenesis. 1997 May;12(3):163-7. doi: 10.1093/mutage/12.3.163.
Human lymphocyte cultures have been used for many years for assessing the in vitro clastogenic potential of test substances. In these assays the harvest time should be based on the cell cycle time in order to ensure that cells are sampled at an appropriate time for the detection of clastogenic effects. The sources of variation in the cell cycle time in routine cytogenetic assays have not been well studied. Consequently 13 laboratories, all members of the Industrial Genotoxicology Group, participated in a collaborative study to measure the variation in cell cycle time in cultured human peripheral blood lymphocytes under various conditions. The study was performed in two phases, spaced 6 months apart. The average generation time (AGT) was measured by the incorporation of bromodeoxyuridine. Very similar AGTs were found in the presence and absence of S9 mix. The mean AGT (mean of four donors) in each laboratory varied from 11.2 to 17.1 h, indicating there is significant variability in cell cycle times of human peripheral blood lymphocytes between laboratories. There was greater variation between laboratories than within laboratories. A comparison of AGT values at 72 h performed in experiments at least 6 months apart indicated good reproducibility in most laboratories. The study indicates that a 24 h post-treatment harvest may result in the analysis of very few first division cells unless very significant cell cycle delay is induced by the test substance. It was also found that a post-harvest time equivalent to 1.5 cell cycles will result in an approximately equal mixture of first and second division cells and therefore should by suitable for assessing both the induction of chromosome aberrations and polyploidy.
多年来，人类淋巴细胞培养一直被用于评估受试物质的体外致断裂潜力。在这些检测中，收获时间应基于细胞周期时间，以确保在适当的时间对细胞进行采样，以便检测致断裂效应。常规细胞遗传学检测中细胞周期时间的变异来源尚未得到充分研究。因此，工业遗传毒理学小组的所有13个实验室参与了一项合作研究，以测量在各种条件下培养的人外周血淋巴细胞的细胞周期时间变异。该研究分两个阶段进行，间隔6个月。通过掺入溴脱氧尿苷来测量平均世代时间（AGT）。在有和没有S9混合物的情况下发现了非常相似的AGT。每个实验室的平均AGT（四个供体的平均值）在11.2至17.1小时之间变化，表明各实验室之间人外周血淋巴细胞的细胞周期时间存在显著差异。实验室之间的差异大于实验室内部的差异。对至少间隔6个月进行的实验中72小时的AGT值进行比较表明，大多数实验室具有良好的重现性。该研究表明，除非受试物质诱导非常显著的细胞周期延迟，否则处理后24小时收获可能导致分析的首次分裂细胞非常少。还发现收获后时间相当于1.5个细胞周期将导致首次和第二次分裂细胞的大致相等混合，因此应该适合于评估染色体畸变和多倍体的诱导。
