MacGregor J T, Wehr C M, Henika P R, Shelby M D
Food Safety Research Unit, Western Regional Research Center, U.S. Department of Agriculture, Albany, California 94710.
Fundam Appl Toxicol. 1990 Apr;14(3):513-22. doi: 10.1016/0272-0590(90)90255-i.
The mouse erythrocyte micronucleus assay has been traditionally carried out using one or two exposures to the test agent, followed by sampling at two or three postexposure times to obtain a sample near the time of the transient peak of micronucleated polychromatic erythrocytes (PCEs). We have demonstrated that frequencies of micronucleated RNA-positive (PCEs) and RNA-negative erythrocytes in blood and bone marrow come to steady state during "continuous" exposure via diet or drinking water, or during repeated daily exposures to test agents by ip injection, gavage, or inhalation. Under these exposure conditions, frequencies of micronucleated cells in peripheral blood approached steady state within 2-3 days in RNA-positive erythrocytes and in 5-6 weeks in RNA-negative erythrocytes. With exposure durations of 6 days (monocrotaline or Crotalaria seeds in diet), 10 days (triethylenemelamine, mitomycin C, 7,12-dimethylbenzanthracene, or colchicine, ip daily), 90 days (triethylenemelamine or urethan in drinking water or 1,3-butadiene via inhalation), or 2 years (benezene by daily gavage), frequencies of micronucleated cells attained and remained at steady state for prolonged periods. At steady state, frequencies of micronucleated RNA-positive cells in bone marrow samples were similar to those in RNA-positive and RNA-negative cells in peripheral blood (e.g., triethylenemelamine in drinking water at 4 micrograms/ml resulted in frequencies of micronucleated RNA-negative erythrocytes in peripheral blood of 27/1000 after 45 days of exposure and 24/1000 after 90 days, with a frequency of 28/1000 in bone marrow RNA-positive erythrocytes after 90 days). The data suggest that the efficiency of the assay would be markedly improved by using a repeated dose schedule with a single sample taken at steady state, rather than scoring multiple samples at various times after a single dose. This approach allows the frequency of micronucleated cells to be measured in a sample of bone marrow or blood obtained at almost any stage of routine toxicity testing.
传统上，小鼠红细胞微核试验是通过对受试物进行一或两次暴露，然后在暴露后的两个或三个时间点取样，以在微核多染红细胞（PCE）短暂峰值出现的时间附近获取样本。我们已经证明，在通过饮食或饮用水进行“持续”暴露期间，或在每天通过腹腔注射、灌胃或吸入重复给予受试物期间，血液和骨髓中微核RNA阳性（PCE）和RNA阴性红细胞的频率会达到稳定状态。在这些暴露条件下，外周血中微核细胞的频率在RNA阳性红细胞中2 - 3天内接近稳定状态，在RNA阴性红细胞中5 - 6周内接近稳定状态。暴露持续时间为6天（饮食中含有野百合碱或猪屎豆种子）、10天（每天腹腔注射三亚乙基三聚氰胺、丝裂霉素C、7,12 - 二甲基苯并蒽或秋水仙碱）、90天（饮用水中含有三亚乙基三聚氰胺或乌拉坦，或通过吸入1,3 - 丁二烯）或2年（每天灌胃给予苯）时，微核细胞的频率会达到并在较长时间内保持稳定状态。在稳定状态下，骨髓样本中微核RNA阳性细胞的频率与外周血中RNA阳性和RNA阴性细胞的频率相似（例如，饮用水中4微克/毫升的三亚乙基三聚氰胺在暴露45天后，外周血中微核RNA阴性红细胞的频率为27/1000，90天后为24/1000，90天后骨髓RNA阳性红细胞中微核频率为28/1000）。数据表明，通过采用重复给药方案并在稳定状态下采集单个样本，而不是在单次给药后的不同时间对多个样本进行评分，该试验的效率将得到显著提高。这种方法允许在常规毒性试验的几乎任何阶段获取的骨髓或血液样本中测量微核细胞的频率。
